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P B Tracy

Publications and source records attributed to P B Tracy.

At least 19 recordsLinked to original sources
PubMed1995

The mechanism of inactivation of human platelet factor Va from normal and activated protein C-resistant individuals.

The inactivation of human platelet factor Va by activated protein C (APC) was analyzed by functional assessment of cofactor activity and Western blotting analysis to visualize the factor Va fragments accompanying proteolysis. Platelets were treated with thrombin to facilitate both their activation as well as the release and further activation of platelet factor Va, followed by APC addition. The rates of inactivation were donor-dependent such that 15-60% of the initial cofactor activity was lost within 5 min of APC addition with as much as 10-20% of the activity still remaining after 2 h of incubation. Western blot analysis using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of the factor V molecule suggested that the factor Va activity resistant to APC inactivation was due to residual heavy chain. Furthermore, in contrast to studies with normal plasma-derived factor Va, two possible cleavage mechanisms could explain the platelet factor Va fragments observed. APC can cleave platelet factor Va initially at Arg506, with subsequent cleavages occurring at Arg306 and Arg679. Alternatively, APC can cleave at Arg306 initially, with further cleavage at Arg679 then at Arg506 or at Arg506 followed by cleavage at Arg679. Similar results were obtained if platelets were removed from the inactivation mixtures and phospholipid vesicles were used to supply the membrane surface required for inactivation, suggesting that the order of platelet factor Va peptide bond cleavage or the amount of cofactor activity remaining was not altered by either of these surfaces. Thus, APC is unable to effect the complete inactivation of platelet factor Va, even though it would appear that the same cleavages which render the plasma cofactor inactive are occurring in the platelet cofactor. Analogous protocols were used to study an individual heterozygous for the Arg506-->Gln506 mutation (Factor V Leiden, Factor VR506Q). With respect to the mutant platelet factor Va in the presence of APC, > 70% of the initial cofactor activity remained after 1 min, with 30% activity still remaining after 2 h. As seen in studies of the APC-catalyzed inactivation of plasma factor VaR506Q, proteolysis of the mutant platelet factor Va confirms that even though cleavage at Arg306 will occur in the absence of cleavage at Arg506, the rate of inactivation is slower. Collectively these data suggest that when compared to normal plasma factor Va, differences in normal platelet factor Va which define: 1) whether the heavy chain is susceptible to cleavage at Arg306 or Arg506 and 2) the extent to which it is cleaved initially at Arg306, in contrast to cleavage of Arg506, will define both the extent and rate of inactivation.

Amino Acid Sequence↗
PubMed1995

Human coagulation factor V is activated to the functional cofactor by elastase and cathepsin G expressed at the monocyte surface.

The ability of intact peripheral blood monocytes to modulate factor V procoagulant activity was studied using electrophoretic and autoradiographic techniques coupled to functional assessment of cofactor activity. Incubation of plasma concentrations of factor V with monocytes (5 x 10(6)/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (> or = 200 kDa) to yield peptides of 150, 140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of cofactor activity. The cleavage pattern observed differed from that obtained with either thrombin or factor Xa as an activator. The incubation time required to achieve full cofactor activity was dependent on the monocyte donor and ranged from 10 min to 1 h and was consistently slightly lower than that obtained with thrombin-activated factor Va. Cofactor activity was not diminished by additional incubation. The cofactor activity generated bound to the monocyte such that a competent prothrombinase complex was formed at the monocyte membrane surface. Furthermore, within 5 min of factor V addition to monocytes, near maximal cofactor activity (approximately 70%) was bound and expressed on the monocyte membrane. The proteolytic activity toward factor V was associated primarily with the monocyte membrane, as little proteolytic activity was released into the cell-free supernatant. Proteolytic activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained with alpha 1-antiproteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin suggested membrane-bound forms of elastase and cathepsin G were mediating, in large part, the proteolysis observed. These data were confirmed using purified preparations of both proteases and a specific anti-human leukocyte elastase antibody. Thus, expression of these proteases at the monocyte surface may contribute to thrombin generation at extravascular tissue sites by catalyzing the activation of the essential cofactor, factor Va, which binds to the monocyte surface and supports the factor Xa-catalyzed activation of prothrombin.

Cathepsin G↗
PubMed1992

Factor Xa interacts with two sites on monocytes with different functional activities.

Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa-catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

Antibodies, Monoclonal↗
PubMed1992

The thrombin high-affinity binding site on platelets is a negative regulator of thrombin-induced platelet activation. Structure-function studies using two mutant thrombins, Quick I and Quick II.

To elucidate the thrombin domains required for high-affinity binding and platelet activation, the platelet binding properties of thrombin and two mutant thrombins, thrombin Quick I and Quick II, were compared to their agonist effects in elevating intraplatelet [Ca2+]. In Quick I, a mutation within the fibrinogen binding groove results in decreased clotting and platelet aggregating activities, whereas in Quick II, a mutation in the primary substrate binding pocket abolishes both activities. Dysthrombin binding was decreased compared to thrombin. The fibrinogen binding groove appeared more important than the primary substrate pocket for high-affinity binding since Quick I showed drastically reduced, and Quick II only slightly reduced, binding affinity (Kd approximately 200 and approximately 10 nM, respectively). The deduced interaction of thrombin with its high-affinity binding site indicated that the thrombin catalytic site is directed toward the platelet surface and therefore, when bound, is proteolytically inactive. Quick I (0.5-5 nM) elicited intraplatelet [Ca2+] fluxes at concentrations where high-affinity binding was undetectable. Saturation of high-affinity binding sites with active-site-modified thrombin did not affect thrombin-induced (0.5 nM) or Quick I-induced (5 nM) responses. In contrast, addition of D-Phe-Pro-Arg chloromethyl ketone (FPRCK) subsequent to thrombin or Quick I stimulation of platelets abolished agonist-induced responses. Since Quick I was only 10-17% as effective as thrombin in increasing intraplatelet [Ca2+], our data support a model in which thrombin acts enzymatically on a platelet membrane "substrate", through an interaction mediated in part by the fibrinogen binding groove of thrombin. This conclusion is consistent with the inhibition observed with high concentrations (greater than 100 nM) of Quick II and FPRCK-modified thrombin (FPR-thrombin) in platelets stimulated with low concentrations of thrombin (less than 0.5 nM) or Quick I (less than 2 nM), consistent with inhibition by substrate depletion. In contrast, concentrations of FPR-thrombin or Quick II (less than 100 nM), which saturated predominantly the high-affinity binding sites, enhanced the platelet responses induced by thrombin (less than 0.5 nM). Thus, occupation of the high-affinity sites with inactive thrombin increased the concentration of active thrombin available for substrate interaction. Quick I-induced responses were not enhanced, consistent with its inability to interact with the high-affinity site. Since thrombin bound to the high-affinity site is proteolytically inactive, we hypothesize that the thrombin high-affinity binding site on platelets functions to alter thrombin activity and platelet activation.

Amino Acid Chloromethyl Ketones↗
PubMed1992

Endotoxin enhances the expression of monocyte prothrombinase activity.

Thrombin is generated on the surface of mononuclear cells (MNCs) through the assembly and function of the prothrombinase complex consisting of the enzyme factor Xa, the cofactor/factor Va, calcium ions, and an appropriate membrane surface for proper assembly of the protein constituents. Assays performed in the presence of factors Va and Xa indicated that endotoxin significantly enhanced the prothrombinase activity (1.5- to 2.5-fold; P less than .001) expressed by MNCs in a dose- and time-dependent manner. Monocytes present in the MNC suspensions were responsible for this increased activity through processes resulting in both enhanced cellular activity and the enhanced release of membranous vesicles. Endotoxin was without effect on the expression of lymphocyte prothrombinase activity. Scanning electron microscopy techniques indicated that endotoxin resulted in extensive membrane blebbing of the monocytes present in the MNC suspensions with no effect on the morphology of the lymphocytes. Within 5 hours, endotoxin maximally enhanced the prothrombinase activity expressed by the monocyte membrane surface 2.8-fold, whereas 8 hours was required to maximally enhance the activity associated with the released vesicles by twofold. The observed increase in activity expressed by the monocyte membrane surface was due solely to endotoxin, since the activity expressed by the unstimulated monocyte membrane surface remained unaltered over time. In contrast, cell vesiculation, which occurred in the absence of any stimulus, was further enhanced by endotoxin. The increase in activity associated with the released vesicles from both stimulated and unstimulated cells paralleled an increase in the vesicle number as determined by flow cytometric analyses. The vesicle released from both unstimulated and stimulated monocytes were indistinguishable in size as determined by image analysis and ranged between 0.05 and 0.3 microns in diameter. 2-Deoxy-D-glucose (2DG) significantly enhanced the prothrombinase activity expressed by the monocyte membrane surface, as well as the released vesicle fraction, when used alone or in addition to endotoxin. The enhanced activity associated with the vesicle fraction again was attributed to the release of more vesicles. In contrast, cycloheximide decreased the prothrombinase activity expressed by the monocyte membrane surface, as well as the activity associated with vesicles released from both stimulated and unstimulated cells. These data suggest that the expression of monocyte prothrombinase activity can be significantly enhanced by endotoxin through processes that alter the monocyte membrane surface and augment the vesiculation process. Both processes appear to be regulated by protein synthesis and adenosine triphosphate (ATP)-dependent mechanisms.

Blood Proteins↗
PubMed1992

Platelet factor Xa receptor.

The assembly and function of the prothrombinase complex on the bovine and human platelet membrane is mediated through binding interactions in which factor Va bound to the platelet surface forms at least part of the "receptor" for factor Xa in a 1:1 stoichiometric complex. A model depicting these binding interactions is shown in Fig. 12. Data from our laboratory indicate that the prothrombinase catalyst assembles in an analogous manner on the surface of monocytes, lymphocytes, neutrophils, and well-defined phospholipid vesicles employed in model systems. The 74,000-Da subunit of factor Va, component E, which mediates the binding of factor Va to either bovine platelets, human monocytes, or phospholipid vesicles, is shown binding to the cell membrane through its putative "receptor." The 94,000-Da subunit of factor Va, component D, is associated with the membrane surface through its metal ion-dependent interaction with component E. Factor Va forms at least part of the receptor that mediates the binding of factor Xa to an appropriate membrane surface, because component E has been shown to contribute significantly to the interaction of factor Xa with either the platelet, monocyte, or vesicle membrane surface. Our data do not preclude the possibility that component D contributes to the binding of factor Xa and the function of the prothrombinase complex. Component D appears to be important for several reasons. Cleavage of component D by activated protein C results in the complete loss of factor Va cofactor activity. An interaction between factor Xa and component D is implied from the observation that factor Xa protects factor Va from activated protein C inactivation. Furthermore, the binding of factor Xa to platelet-bound factor Va results in the time-dependent cleavage of components D and D'. Because component D is not required absolutely for prothrombinase complex assembly, we would speculate that it may be important in mediating prothrombin binding (depicted as a three-domain molecule) and increasing the catalytic efficiency of the enzymatic complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

A case of acquired factor X deficiency with in vivo and in vitro evidence of inhibitor activity directed against factor X.

A 67-year-old woman had symptoms of an upper respiratory tract infection for which she received a five-day course of erythromycin. Epistaxis and gross hematuria subsequently developed, and the patient was found to have a selective Factor X deficiency. She received supportive therapy and prothrombin complex concentrates (Factors II, VII, IX, and X), with subsequent resolution of her transient Factor X deficiency. Her hospital course, however, was complicated by the development of multiple cerebral infarctions. This is the tenth reported case of transient Factor X deficiency not associated with amyloidosis. In seven of the previous cases, as in this patient, the deficiency was associated with a preceding upper respiratory infection. This is the only case, however, with evidence of inhibitory activity in the plasma that was directed toward Factor X.

Aged↗
PubMed1990

Effects of nutritional folate deficiency on the adhesive properties of murine melanoma cells.

Folate deficient murine B16 melanoma cells adhered more rapidly and in higher percentages to plastic plates or dishes coated with laminin or fibronectin than folate replete cells. These changes in the adhesive properties of murine melanoma cells induced by nutritional folate deficiency were not mediated by changes in cell size, proliferative capacity or cell cycle distribution. While melanoma cells served as a suitable surface for prothrombinase complex formation, folate deficiency did not alter this membrane function, suggesting that the membrane changes associated with folate deficiency are relatively specific.

Animals↗
PubMed1990

Functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin.

The functional characterization of human platelet-released factor V and its activation by factor Xa and thrombin was studied by functional assessment of cofactor activity and Western blotting analyses of platelet releasates, obtained by stimulating washed suspensions of platelets with various agonists, including collagen, collagen with ADP, and the calcium ionophore A23187. Platelet factor V was released as a partially proteolyzed molecule that was bound to platelet microparticles, irrespective of the agonist used. Radiolabeled plasma factor V was not cleaved for up to 30 min following release when added to platelets prior to stimulation, suggesting that platelet factor V was stored in a partially proteolyzed form. Released platelet factor V possessed significant cofactor activity that was increased only 2-3-fold by either factor Xa or thrombin. The factor V subunits that expressed cofactor activity were isolated and found to consist of peptides of Mr = 220,000 and 150,000. Incubation of released platelet factor V with factor Xa or thrombin yielded the same cleavage pattern, in which two peptides of Mr = 105,000 and 74,000 appeared to be electrophoretically indistinguishable from thrombin-activated plasma factor V. Under the conditions of these studies, factor Xa activated platelet-released factor V 50-100 times more effectively than thrombin. This observation may be due in part to the existence of platelet factor V in a partially proteolyzed state, or its association with platelet microparticles following platelet stimulation. These data collectively suggest that platelet-released factor V may be the foremost initiator of prothrombinase complex assembly and function during the early stages of coagulation with additional cofactor activation accomplished by factor Xa.

Adenosine Diphosphate↗
PubMed1990

Activation of human factor V by factor Xa and thrombin.

The activation of human factor V by factor Xa and thrombin was studied by functional assessment of cofactor activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by either autoradiography of 125I-labeled factor V activation products or Western blot analyses of unlabeled factor V activation products. Cofactor activity was measured by the ability of the factor V/Va peptides to support the activation of prothrombin. The factor Xa catalyzed cleavage of factor V was observed to be time, phospholipid, and calcium ion dependent, yielding a cofactor with activity equal to that of thrombin-activated factor V (factor Va). The cleavage pattern differed markedly from the one observed in the bovine system. The factor Xa activated factor V subunits expressing cofactor activity were isolated and found to consist of peptides of Mr 220,000 and 105,000. Although thrombin cleaved the Mr 220,000 peptide to yield peptides previously shown to be products of thrombin activation, cofactor activity did not increase. N-Terminal sequence analysis confirmed that both factor Xa and thrombin cleave factor V at the same bond to generate the Mr 220,000 peptide. The factor Xa dependent functional assessment of 125I-labeled factor V coupled with densitometric analyses of the cleavage products indicated that the cofactor activity of factor Xa activated factor V closely paralleled the appearance of the Mr 220,000 peptide. This observation facilitated the study of the kinetics of factor V activation by allowing the activation of factor V to be monitored by the appearance of the Mr 220,000 peptide (factor Xa activation) or the Mr 105,000 peptide (thrombin activation). Factor Xa catalyzed activation of factor V obeyed Michaelis-Menten kinetics and was characterized by a Km of 10.4 nM, a kcat of 2.6 min-1, and a catalytic efficiency (kcat/Km) of 4.14 X 10(6) M-1 s-1. The thrombin-catalyzed activation of factor V was characterized by a Km of 71.7 nM, a kcat of 14.0 min-1, and a catalytic efficiency of 3.26 X 10(6) M-1 s-1. This indicates that factor Xa is as efficient an enzyme toward factor V as thrombin.

Amino Acid Sequence↗
PubMed1988

Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection.

Kinetic analyses were done to determine what effect factor Xa and protein S had on the activated protein C (APC)-catalyzed inactivation of factor Va bound to phospholipid vesicles or human platelets. In the presence of optimal concentrations of phospholipid vesicles and Ca2+, a Km of 19.7 +/- 0.6 nM factor Va and a kcat of 23.7 +/- 10 mol of factor Va inactivated/mol of APC/min were obtained. Added purified plasma protein S increased the maximal rate of factor Va inactivation only 2-fold without effect on the Km. Protein S effect was unaltered when the phospholipid concentration was varied by 2 orders of magnitude. The reaction on unactivated human platelets yielded a Km = 12.5 +/- 2.6 nM and kcat = 6.2 +/- 0.6 mol of factor Va inactivated/mol of APC/min. Added purified plasma protein S or release of platelet protein S by platelet activation doubled the kcat value without affecting the Km. Addition of a neutralizing anti-protein S antibody abrogated the effect of plasma protein S or platelet-released protein S, but was without effect in the absence of plasma protein S or platelet activation. Studies with factor Xa indicated that factor Xa protects factor Va from APC-catalyzed inactivation by lowering the effective concentration of factor Va available to interact with APC. From these data a dissociation constant of less than 0.5 nM was calculated for the interaction of factor Xa with membrane-bound factor Va. Protein S abrogated the ability of factor Xa to protect factor Va from inactivation by APC without affecting the interaction of factor Xa with factor Va. These combined data suggest that one physiological function of protein S is to allow the APC-catalyzed inactivation of factor Va in the presence of factor Xa.

Blood Platelets↗
PubMed1988

Regulation of thrombin generation at cell surfaces.

A complex series of reactions are involved in the assembly, function, and regulation of the prothrombinase complex. Since the enzyme is multicomponent in nature and each component is required for catalytic function, modulation of enzymatic activity can be achieved in a variety of ways. In addition, since complex assembly so profoundly affects reaction rates, mechanisms that perturb complex formation either positively or negatively have a profound effect on thrombin generation and its local physiologic effects. All of the cells that support prothrombinase assembly and hence thrombin generation respond to thrombin in a variety of ways. Thrombin selectively binds to thrombomodulin and heparin-like molecules expressed on the endothelial cell surface. Thrombin induces the release (and possible synthesis of) prostacyclin, plasminogen activator inhibitor, platelet-derived growth factor, and interleukin-1 and inhibits the release of plasminogen activator from vascular endothelium. Interleukin-1 is a potent mediator of inflammatory phenomena as well as an inducer of tissue factor synthesis in vascular endothelium. With respect to platelets, thrombin selectively binds and stimulates the platelet release reaction and subsequent aggregation. The thrombin-induced release of platelet-derived growth factor from both platelets and vascular endothelium may play a role in inflammation, wound healing, and atherogenesis. Thrombin itself is a potent mitogen of mesenchymal cells, and more recently has been shown to be not only a chemoattractant, but also a mitogen for monocytes. Thrombin also appears to bind selectively to monocytes and in so doing induces release of interleukin-1. Thrombin affects a myriad of cellular responses related to hemostasis, thrombosis, inflammation, would repair, and atherogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Enhanced thrombin generation and platelet binding on herpes simplex virus-infected endothelium.

Atherosclerotic lesions have been reported to contain herpes simplex virus 1 (HSV-1) genomic material. This, and other previous evidence, suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV-1 lesions manifest marked fibrin deposition in microvessels. In this report we show that very early infection of human endothelial cells with HSV-1 appears to alter surface conformation as detected by merocyanine 540 staining. Concomitantly, the efficiency of prothrombinase complex assembly increases, resulting in a 2- to 3-fold accelerated rate of thrombin generation on the cell surface. Increased thrombin generation is probably doubly procoagulant, since we also demonstrate that thrombin-induced platelet accumulation on HSV-infected endothelium (50.7 +/- 9.3%) is increased compared to uninfected endothelium (9.5 +/- 2.1%; P less than 0.002). Associated with HSV infection, prostacyclin secretion in response to thrombin is diminished by a factor of 20, probably explaining the enhanced platelet attachment. We conclude that HSV infection shifts endothelial cell properties from anticoagulant to procoagulant, both by promoting prothrombinase complex formation and function and by increasing platelet binding, well before cell disruption takes place. Virus-induced changes in the endothelial plasma membrane and diminished prostacyclin secretion are suggested as the pathways for this pathophysiologic mechanism, which may be germane to atherosclerotic thrombosis as well as HSV-mediated tissue necrosis.

Arteriosclerosis↗
PubMed1987

Abnormal formation of the prothrombinase complex: factor V deficiency and related disorders.

A membrane-bound, Ca2-dependent complex of the cofactor factor Va and the enzyme factor Xa comprises the prothrombinase coagulation complex, which catalyzes the proteolytic conversion of prothrombin to thrombin. In normal hemostasis, the platelet is presumed to supply the surface membrane and thus constitutes the site at which an enzymatically functional complex assembles and thrombin generation occurs. Factor Va, the two subunit protein produced by thrombin activation of factor V, is an essential, nonenzymatic cofactor of the prothrombinase complex. Factor Va performs its cofactor role in part by binding to the platelet membrane and functioning as the membrane receptor for factor Xa in a 1:1 stoichiometric complex of high affinity (Kd = 10(-10) M). Factor Va also appears to participate in the binding of prothrombin to the enzymatic complex. Because deletion of factor Va from the prothrombinase complex decreases the rate of thrombin generation by four orders of magnitude, the essential role it plays is easily understood. Therefore, in the evaluation of factor Va function in the prothrombinase complex, the ability of factor Va to support various binding interactions with the platelet, factor Xa, and prothrombin must be considered. Factor Va can be made available from two potential blood compartments: the plasma and platelets. Approximately 80 per cent of the total blood factor V circulates in plasma whereas the remaining 20 per cent is contained within platelet granules. The relative contribution of plasma versus platelet factor V to factor Va binding interactions in the prothrombinase complex are not clearly defined. However, data from our laboratory and several others suggest that factor V stored and released from platelets is of utmost importance in maintaining normal hemostasis. A discussion of these data relative to congenital and acquired deficiencies of both plasma and platelet factor V is the subject of this report.

Blood Platelets↗
PubMed1987

The isolation of human platelet factor V.

Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

Blood Coagulation↗
PubMed1985

Human prothrombinase complex assembly and function on isolated peripheral blood cell populations.

A membrane-bound Ca2+-dependent complex of the cofactor Factor Va and the enzyme Factor Xa comprises the prothrombinase coagulation complex which catalyzes the proteolytic conversion of prothrombin to thrombin. Analyses of the kinetics of prothrombin activation permit calculation of the stoichiometry and binding parameters governing the functional interactions of Factor Va and Factor Xa with isolated thrombin-activated human platelets and isolated leukocyte subpopulations. Our kinetic approach indicates that Factor Xa binds to approximately 2700 +/- 1000 (n = 8) functional sites on the surface of thrombin-activated platelets with an apparent dissociation constant (Kd) equal to 1.18 +/- 0.53 X 10(-10) M and kcat equal to 19 +/- 7 mol of thrombin/s/mol of Factor Xa bound. The store of Factor V in normal platelets prevents an analogous determination of the functional Factor Va platelet binding sites. Factor Va and Factor Xa titrations performed using platelets from a Factor V antigen-deficient individual indicate that Factor Va and Factor Xa form a 1:1 stoichiometric complex on the surface of thrombin-activated platelets. Both binding isotherms are governed by the same apparent Kd (approximately equal to 10(-10) M) and expressed the same kcat/site (14-17 s-1. Factor Xa-platelet binding parameters are not altered by the use of different platelet agonists, the choice of anticoagulant, or platelet washing procedure. Kinetics of prothrombin activation indicate also that monocytes, lymphocytes, and neutrophils possess, respectively, 16,000, 45,000, and 8,000 Factor Va-Factor Xa receptor sites/cell, which are all governed by apparent KdS approximately equal to 10(-10) M. Enzymatic complexes bound to monocytes or neutrophils exhibit kcat values similar to the platelet-bound complex. Complexes bound to lymphocytes are only 25% as active.

Adult↗
PubMed1985

Tumor cell generation of thrombin via functional prothrombinase assembly.

Prothrombinase affects the proteolytic conversion of prothrombin to thrombin and is the penultimate enzyme in the common coagulation pathway. Prothrombinase is a complex in which the proteinase, Factor Xa, a cofactor, Factor Va, and calcium are bound to a membrane surface to generate the active enzyme. Guinea pig line 1 and line 10 tumor cells, grown as primary cultures from ascites tumors or as cell lines in culture, provide a surface that interacts with coagulation Factor Va and Xa and with calcium ions to form this enzyme complex. Cultured human colorectal carcinoma cells (Colo 205) also participate in prothrombinase complex assembly and function. Prothrombinase generation was measured by following the kinetics of prothrombin conversion to thrombin. Thrombin generation was monitored continuously using the reversible thrombin inhibitor, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide, which displays enhanced fluorescence upon binding to thrombin. Analyses of kinetic data indicate that the apparent dissociation constants (1-4 X 10(-10) mol/liter) and the number of Factor Va-Xa binding sites per tumor cell are comparable to values reported for human and bovine platelets, human lymphocytes, and monocytes. Guinea pig lymphocytes were also active, while erythrocytes were inactive, in the prothrombinase assay. Membrane vesicles, shed by guinea pig and human tumor cells into conditioned medium, also supported functional prothrombinase activity. Although earlier studies indicated that tumor cells may initiate coagulation, this is the first demonstration that tumor cells are competent to bring clotting to fruition by generating thrombin, a step essential to fibrin generation. These data suggest that tumor cells, in the presence of clotting initiators and appropriate coagulation factors, are sufficient to generate the fibrin deposited in solid tumors.

Animals↗