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PubMed1987

Catalysis of thrombin inhibition provides an index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.

Previous studies have demonstrated that standard anticoagulant tests are poor indices of the antithrombotic potential of glycosaminoglycans which are weak catalysts of the thrombin-antithrombin III reaction. In this study we investigated whether the catalysis of thrombin inhibition by plasma could serve as a reliable index for assessing the antithrombotic effectiveness of glycosaminoglycans. Equal volumes of 125I-thrombin and control or test plasma were incubated for up to 10 min at 37 degrees C. Inactivation of thrombin was then determined after 7.9% SDS-polyacrylamide gel electrophoresis and subsequent autoradiography. Increasing concentrations of heparin (greater than 0.066 micrograms/mL or 0.01 USP units/mL) and dermatan sulfate (greater than 0.1 micrograms/mL) could be readily demonstrated in undiluted plasma by enhanced formation of complexes of thrombin with antithrombin III and heparin cofactor II respectively. However, the detection of any catalytic effect of the two glycosaminoglycans decreased significantly with increasing plasma dilutions. When ex vivo plasmas obtained from rabbits that had been injected with the minimum dose of any one of seven glycosaminoglycans required to achieve their optimal antithrombotic effect were assessed for their ability to catalyse thrombin inhibition, there was approximately a 2-fold increase in the amount of thrombin inactivated 30 s after the thrombin had been added to the plasma. The enhanced inhibition of thrombin was achieved by catalysis of antithrombin III and/or heparin cofactor II activities. These results suggest that measurement of the catalysis of thrombin inactivation in undiluted plasma is a sensitive and reliable index for estimating the antithrombotic potential of glycosaminoglycans in rabbits.

Animals↗
PubMed1988

Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E.

In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.

Amino Acid Sequence↗
PubMed1987

The in situ inhibition of prothrombinase-formed human alpha-thrombin and meizothrombin(des F1) by antithrombin III and heparin.

The inhibition of thrombin by antithrombin III (AT III) and heparin has been studied in pure systems to determine the kinetics of inhibition during human prothrombin activation. The present study shows that prothrombinase-catalyzed prothrombin activation resulted in the generation of thrombin and meizothrombin(des F1). In the absence of heparin the second-order rate constants of the inactivation of both thrombin and meizothrombin(des F1) formed in the reaction mixture appeared to be identical, k = 3.7 X 10(5) M-1 min-1. The rate constant of inhibition of purified thrombin was 6.5 X 10(5) M-1 min-1. In the presence of heparin the decay of the amidolytic activity was biexponential and could be modeled by a four-parameter equation to determine the pseudo first-order rate constants of inhibition as well as the composition of the reaction with respect to the levels of thrombin and meizothrombin(des F1). The ratio of thrombin over meizothrombin(des F1) varied with the initial prothrombin concentration. Heparin catalyzed the AT III inhibition of thrombin but not meizothrombin(des F1) formed during the prothrombin activation. Thrombin, generated by (Xa-Va-phospholipid-Ca2+) was inhibited by AT III/heparin more slowly than purified thrombin, and the saturation kinetics of the inhibition with respect to AT III differed from those found with purified thrombin.

Animals↗
PubMed1987

Unmasking of thrombin vasoconstriction in isolated perfused dog hearts after intracoronary infusion of air embolus.

Effects of intracoronary thrombin and acetylcholine administration on coronary vascular reactivity were studied in isolated perfused (Langendorff) dog hearts before and after air embolus treatment. Under constant flow condition with control coronary perfusion pressure of 71.4 +/- 5.4 mm Hg (mean +/- S.E.M. of eight hearts), infusion of thrombin (0.1 U/ml) resulted in an immediate decrease (-32.8 +/- 2.7 mm Hg) in coronary perfusion pressure, reaching a maximum in approximately 20 to 25 sec. Similar vasodilatory response (-30.5 +/- 1.9 mm Hg, M +/- S.E.M. of six hearts) also was observed with administration of 0.1 microM acetylcholine. Introduction of 0.3 ml air embolus into the coronary circulation before thrombin or acetylcholine testings, however, showed a 100 and 63% decrease in their respective coronary vasodilatory responses. More importantly, infusion of thrombin into these previously air embolus-treated hearts actually resulted in a potent vasoconstriction by increasing the coronary perfusion pressure (+48.2 +/- 5.7 mm Hg, mean +/- S.E.M. of six hearts) above the control level. The coronary vasoconstrictory effect of thrombin was reversible upon cessation of thrombin infusion or with concurrent administration of sodium nitroprusside (1 microM). Similarly, synthetic thrombin inhibitor phenylalanyl-prolyl-arginine chloromethyl ketone (0.1 microM) also was capable of both inhibiting and reversing thrombin-induced vasoconstriction. The present finding of a potent thrombin-induced coronary vasoconstriction in intact hearts after air embolus infusion further supports the hypothesis that an activation of thrombin in certain pathological states with injured endothelium may have an important role in the genesis of coronary vasospasm.

Acetylcholine↗
PubMed1986

Injury to cultured endothelial cells by thrombin-stimulated platelets.

In vivo, stimulated platelets may injure the endothelium. We have used cultured endothelial cells to assess endothelial cell damage caused by platelet stimulation with thrombin. Endothelial cells were cultured from umbilical veins and semiconfluent cultures were labeled with Na2 51CrO4. Twenty four hours later washed human platelets (final concentration 200,000 platelets/microliters) and thrombin (final concentration 4 units/ml) were added to the medium and the culture dish was shaken for 15 minutes. The percentage of cells detached from the culture dish and the percentage of 51Cr lost from the endothelial cells into the ambient fluid during the shaking were determined and used as indicators of cell injury. Increased percentages of loosened cells and 51Cr in the ambient fluid were observed with platelet suspension and thrombin compared to controls with neither platelet suspension nor thrombin and controls with either platelet suspension or thrombin. The platelet-free supernatant obtained after reaction of the platelets with thrombin also increased the percentage of loosened cells, but it did not increase the percentage of 51Cr in the ambient fluid to a significant degree. Thrombin alone caused a moderate loss of 51Cr, but no increased loosening of cells. Treatment of the platelets with acetylsalicylic acid prior to the experiment depressed the detachment effect of thrombin-stimulated platelets, but did not alter the effect on the release of 51Cr into the ambient fluid. Scanning and transmission electron microscopy of cultured endothelial cells exposed to thrombin-stimulated platelets confirmed the presence of loosening and injury to the endothelial cells. Thus, platelet stimulation with thrombin had at least two effects on the cultured endothelial cells: a loosening effect caused by material released from the platelets; an injury effect which, in order to reach its maximum, required the presence of stimulated platelets.

Aspirin↗
PubMed1979

[Activation of the anticoagulation system following intravenous administration of beta-thrombin].

Beta-thrombin possessing high esterase activity and tracing coagulating ability, being product of limited proteolysis of alpha-thrombin in vitro, accelerates recalcification time and thrombin generation in plasma, but not the conversion of prothrombin to enzyme. Thus, beta-thrombin is the activator of early stages of blood coagulation, does not possess fibrinolytic activity and does not activate plasminogen. The i. v. administration of beta-thrombin to rats induces changes in blood coagulability which are accompanied by an increase in plasma recalcification time, total fibrinolytic activity and non-enzymatic fibrinolysis. Nothing of the kind occurs after administration alpha-thrombin, having tracing clotting activity similar to R-thrombin activity. The data obtained suggest the possibility of reflex activation of the anticoagulating system by beta-thrombin or undirectly by alpha-thrombin generated by beta-thrombin activation at early stages of blood coagulation.

Animals↗
PubMed1977

Antithrombin III and heparin inactivation in thrombin involving reactions.

The inhibitory capacity of antithrombin III (AT III) was measured by a quantitative method independent of the velocity of inhibition. When AT III was in excess of thrombin in plasma or in purified system the capacity of inhibitor decreased quantitatively in proportion to the amount of thrombin neutralized. Heparin present in reaction together with thrombin invariably induced a more extensive utilization of inhibitor than thrombin alone. The extent of this additional loss of inhibitory capacity was to a limited degree related to the concentration of heparin. Heparin itself was neutralized in thrombin-AT III reaction losing its anticoagulant property in proportion to the amount of thrombin bound by inhibitor. This quantitative neutralization of heparin occurred not only when the anticoagulant participated in thrombin-AT III binding but also when heparin was added to a medium containing a preformed thrombin-AT III complex. These results suggest that acceleration of binding and increased utilization of binding capacity are the two regular effects of heparin on thrombin-involving reactions of AT III. Both of these effects may be abolished by quantitative binding of heparin to thrombin-AT III complex.

Antithrombins↗
PubMed1984

Heparan sulfate and dermatan sulfate inhibit the generation of thrombin activity in plasma by complementary pathways.

Heparan with a low affinity for antithrombin III has previously been demonstrated to inhibit thrombin generation in both normal plasma and plasma depleted of antithrombin III. In addition, standard heparin and heparin with a low affinity for antithrombin III have been demonstrated to have equivalent inhibitory actions on thrombin generation in plasma depleted of antithrombin III. These observations prompted the investigation of the effects of four normal vessel wall glycosaminoglycans (heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate) on the intrinsic pathway generation of thrombin and factor Xa and on the inactivation of thrombin and factor Xa in plasma. Heparan sulfate inhibited thrombin generation and accelerated the inactivation of added thrombin and factor Xa in normal plasma but not in antithrombin III-depleted plasma. In contrast, dermatan sulfate inhibited thrombin generation in both normal and antithrombin III-depleted plasma. In addition, heparan sulfate was an effective inhibitor of factor Xa generation, while dermatan sulfate was not. Neither chondroitin-4-sulfate nor chondroitin-6-sulfate inhibited the generation of thrombin or factor Xa nor did they accelerate the inactivation of factor Xa or thrombin by plasma. These results suggest that heparan sulfate acts primarily by potentiating antithrombin III, while dermatan sulfate acts by potentiating heparin cofactor II. The inhibition of thrombin generation by heparan sulfate and dermatan sulfate thus appears to occur by complementary pathways, both of which may contribute to the anticoagulation of blood in vivo.

Anticoagulants↗
PubMed1983

Mechanism of thrombin binding to endothelial cells.

The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5'-phosphate (PLP2-thrombin) destroyed the high-affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H-heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

Antithrombin III↗
PubMed1984

Characterization of the thrombin-induced contraction of vascular smooth muscle.

Purified human alpha-thrombin induced a sustained contraction of isolated rabbit aorta and dog coronary arteries. These vascular tissues also exhibited a refractoriness towards a second thrombin exposure. The extent of tachyphylaxis exhibited by the aorta correlated with the initial concentration of thrombin and the length of time the tissue was exposed to thrombin. The thrombin-induced contraction in the aorta was not blocked by phospholipase or cyclooxygenase inhibitors, but it was inhibited in the presence of hirudin, heparin, nitroglycerin, and nitroprusside. Nitroglycerin, nitroprusside, and hirudin also inhibited the contraction in the dog coronary artery. Ca++ channel blockers did not inhibit the thrombin-induced contraction in the coronary artery, although a small inhibition was observed in Ca++-free media. In both tissues, equivalent contractile responses were obtained using equimolar quantities of beta-, tetranitromethane-, and alpha-thrombin, even though the latter's coagulant activity was 30-40 times that of the modified thrombins. However, if the catalytic activity of thrombin was inhibited by modification with Tos-Lys-CH2Cl, hirudin, or heparin/antithrombin III, the vasoconstrictor activity was also lost. These studies suggest that alterations of the fibrinogen-binding site do not affect the contractile activity of thrombin. The contraction may be the result of a proteolytic interaction of the active site of the enzyme with vascular smooth muscle.

Animals↗
PubMed1976

Inactivation of alpha- and beta- thrombin by antithrombin-III and heparin.

Inactivation of alpha- and beta-thrombin by antithrombin-III and heparin was studied, since it had been suggested that two forms of thrombin exist with respect to heparin sensitivity (Machovich 1975b). It was found that the inactivation rates of alpha- and beta-thrombin by antithrombin were different, namely alpha-thrombin was more sensitive to antithrombin than beta-thrombin. Heparin facilitated the complex formation between alpha-thrombin and antithrombin-III, whereas beta-thrombin inactivation was only slightly affected. Furthermore, heparin protected alpha-thrombin against the inactivating effect of heat, while beta-thrombin lost its activity during the heat treatment. These findings suggest that the formation of beta-thrombin in blood circulation may have an important role in thrombosis predisposition.

Alpha-Globulins↗
PubMed1994

alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood.

Previous studies have shown that thrombin binds to the extracellular endothelial matrix and remains biologically active. In the present study, the role of matrix-bound alpha-thrombin in thrombus formation was investigated by utilizing a model system of thrombogenesis. Plastic cover-slips coated with either matrix-bound alpha-thrombin or matrix-bound active site inactivated thrombin (DIP-alpha-thrombin) were positioned in parallel-plate perfusion chambers and subsequently exposed to non-anticoagulated human blood at a venous wall shear rate of 100/s. The blood was drawn directly from an antecubital vein by a roller pump placed distally to the perfusion chamber. The thrombotic deposits on the matrix, fibrin deposition and platelet thrombus volume, were morphologically evaluated. Matrix-bound alpha-thrombin enhanced the fibrin deposition and thrombus volume on matrices of non-stimulated endothelium with 91% (P < 0.001) and 94% (P < 0.05), respectively. In contrast, binding of DIP-alpha-thrombin to matrices of stimulated endothelium reduced the fibrin deposition by 33% (P < 0.05), but had no effect on the platelet thrombus volume. Translocation of thrombin molecules from upstream matrix areas to binding sites farther downstream on the matrix was indicated in experiments with matrices of stimulated endothelium, which showed enhanced fibrin deposition on downstream areas. Our findings are compatible with a prominent role for matrix-bound alpha-thrombin in thrombogenesis, and in particular on endothelial matrices without tissue factor. The role of matrix-bound alpha-thrombin on tissue factor containing matrices appears less prominent, although it is significant.

Blood Coagulation↗
PubMed1994

Biochemical effect and kinetics of thrombin inhibition by GYKI-14766.

The tripeptide aldehyde GYKI-14766 (D-MePhe-Pro-Arg-H) synthesized by Bajusz et al. in 1975 is a specific, reversible thrombin inhibitor. It was found effective in vitro in clotting time assays as well as in vivo in thrombosis models. To study the biochemical effects of the inhibitor various experimental setups were applied. First we measured the binding of thrombin to platelets using 125J-thrombin. KD was 55 nM. Second, 125J-thrombin was displaced by thrombin or by a GYKI-14766-thrombin-complex with similar efficacy. However, the binding of thrombin to the platelets increased the intracellular free Ca2+ concentration, but the inhibitor-thrombin complex did not influence it. Analyzing the kinetics of the reactions involved we found that the formation of the GYKI-14766-thrombin complex was slower than the triggering of the platelet Ca2+ signal by thrombin.

Animals↗
PubMed1994

An autoantibody directed against human thrombin anion-binding exosite in a patient with arterial thrombosis: effects on platelets, endothelial cells, and protein C activation.

An autoantibody, developed by a patient with severe and recurrent arterial thrombosis, was characterized to be directed against the anion-binding exosite of thrombin, and inhibited all thrombin interactions requiring this secondary binding site without interfering with the catalytic site. The effect of the antibody was studied on thrombin interactions with platelets and endothelial cells from human umbilical veins (HUVEC). The autoantibody specifically and concentration-dependently inhibited alpha-thrombin-induced platelet activation and prostacyclin (PGI2) synthesis from HUVEC. It had no effect when gamma-thrombin or the thrombin receptor activation peptide SFLLR were the inducers. The effect of the antibody on protein C activation has been studied. The antibody blocked the thrombin-thrombomodulin activation of protein C. The inhibition of the activation was maximal with a low concentration of thrombomodulin. The fact that the autoantibody inhibited concentration-dependent alpha-thrombin-induced platelet and endothelial cell functions emphasizes the crucial role of the anion-binding exosite of thrombin to activate its receptor. In regard to the pathology, the antibody inhibited two vascular processes implicated in thrombin-antithrombotic functions, PGI2 secretion, and protein C activation, which could be implicated in this arterial thrombotic disease.

Anions↗
PubMed1993

Thrombin generation during cardiac surgery: is heparin the ideal anticoagulant?

Blood samples were collected from 43 patients undergoing elective cardiac surgery to determine the extent of thrombin generation and inhibition in patients when receiving heparin while undergoing cardiopulmonary bypass (CPB). Plasma prothrombin fragment F1 + 2 and thrombin-antithrombin III (TAT) levels were measured as markers of thrombin generation and inhibition, respectively. Both F1 + 2 and TAT levels increased significantly during the course of CPB despite the heparin causing significant systemic anticoagulation, i.e. the activated coagulation time (ACT) was prolonged to greater than 400 s throughout the entire surgical procedure. The extent of thrombin generation increased with time on CPB but did not differ between patients receiving normothermic and hypothermic cardioplegia during CPB. Furthermore, thrombin generation increased following the neutralization of the heparin with protamine sulphate, and continued to be elevated significantly 24 h post surgery. The observation that high dose heparin did not prevent thrombin generation during CPB, is consistent with previous experimental studies demonstrating that thrombin bound to fibrin or other surfaces (e.g. the CPB conduit) is resistant to antithrombin III/heparin inhibition, and thus able to facilitate further thrombin generation. The observation that thrombin generation continued to be elevated post surgery i.e. 24 h after neutralizing the heparin with protamine sulphate, suggests that the high dose heparin did not inhibit effectively all of the thrombin that had been generated. Thus, CPB patients may be at risk not only of bleeding and other side-effects associated with the acute use of high dose heparin, but may also be at risk of further thrombosis-related events either acutely or chronically.

Aged↗
PubMed1993

Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin.

The procoagulant alpha-thrombin is produced by the proteolytic cleavages of a minimum of two peptide bonds Arg274-Thr275 and Arg323-Ile324 in prothrombin. The Arg323-Ile324 cleavage is required for the expression of the active site of thrombin (Morita, T., Iwanaga, S. Suzuki, T. (1976) J. Biochem. (Tokyo) 79, 1089-1108; Hibbard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Biochemistry 21, 2285-2292). It is not yet clear to what extent the proteolytic events are responsible for exposing protein recognition exosites on thrombin. We employed high resolution NMR spectroscopy to examine interactions of prothrombin and thrombin with synthetic hirudin peptides targeted toward the fibrinogen recognition exosite of thrombin. The hirudin tail synthetic analogues (acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65/G ly65-OH) exhibited similar NMR relaxation enhancements (line broadening patterns and transferred nuclear Overhauser effects) with human prothrombin as with human alpha-thrombin, indicating that both proteins bind the peptide in a similar manner. The protein-induced relaxation enhancements are specific to the interaction of the hirudin peptides with the fibrinogen recognition exosite of thrombin since no significant effects were observed with either human serum albumin or with human gamma-thrombin, which has an impaired recognition exosite. The binding affinities were determined from NMR relaxation time measurements, which gave approximate Kd values of 500 microM and < 100 microM for prothrombin and alpha-thrombin, respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in alpha-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form.

Amino Acid Sequence↗
PubMed1995

Prolonged expression of procoagulant activity of human platelets degranulated by thrombin.

Platelets are exposed to thrombin when they take part in arterial thrombus formation, and they may return to the circulation when they are freed by fibrinolysis and dislodged by flowing blood. Thrombin causes the expression of procoagulant activity on platelets, and if this activity persists, the recirculating platelets may contribute to subsequent thrombosis. We have developed techniques to degranulate human platelets by treatment with thrombin, and recover then as single, discrete platelets that aggregate in response to both weak and strong agonists. In the present study we examined the duration of procoagulant activity on the surface of thrombin-degranulated platelets by two methods: a prothrombinase assay, and the binding of 125I-labeled annexin. Control platelets generated 0.9 +/- 0.4 U thrombin per 10(7) platelets in 15 min. Suspensions of thrombin-degranulated platelets formed 5.4 +/- 0.1 U thrombin per 10(7) platelets in this time. Binding of 125I-annexin V was also greater with thrombin-treated platelets than with control platelets (controls: 1.7 +/- 0.1 ng annexin/10(7) platelets; thrombin-degranulated platelets: 6.8 +/- 0.2 ng annexin/10(7) platelets). With thrombin-degranulated platelets, increased procoagulant activity and annexin binding persisted for at least 4 h after degranulation and resuspension, indicating that the catalytic activity for the prothrombinase complex is not reversed during this time. These platelets maintained their ability to aggregate for 4 h, even in response to the weak agonist, ADP. Thus, platelets that have taken part in thrombus formation and returned to the circulation may contribute to the promotion of further thrombotic events because of the persistence of procoagulant activity on their surface.

Amino Acid Sequence↗
PubMed1997

Thrombin generation by apoptotic vascular smooth muscle cells.

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Adenovirus E1A Proteins↗