Fibrinogen and prothrombin binding is enhanced to the Pro33 isoform of purified integrin alphaIIbbeta3.
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Blood Platelets↗
Biomedical subjects
P Thiagarajan
Publications and source records attributed to P Thiagarajan.
P-selectin in arterial thrombosis.
P-selectin is a transmembrane protein present in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells. Following activation, it is rapidly translocated to the cell surface. P-selectin expression in platelets has been shown to be elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells as well as interactions of platelets with leukocytes. Platelet P-selectin interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocytes to form platelet-leukocyte aggregates. Furthermore, this interaction of P-selectin with PSGL-1 induces the upregulation of tissue factor, several cytokines in leukocytes and the production of procoagulant microparticles, thereby contributing to a prothrombotic state. P-selectin is also involved in platelet-platelet interactions, i. e. platelet aggregation which is a major factor in arterial thrombosis. P-selectin interacts with platelet sulfatides, thereby stabilizing initial platelet aggregates formed by GPIIb/IIIa-fibrinogen bridges. Inhibtion of the P-selectin-sulfatide interaction leads to a reversal of platelet aggregation. Thus, P-selectin plays a significant role in platelet aggregation and platelet- leukocyte interactions, both important mechanisms in the development of arterial thrombosis.
Animals↗
Sulfatides: targets for anti-phospholipid antibodies.
BACKGROUND: Sulfatides are sulfated glycosphingolipids expressed on the surface of erythrocytes, leukocytes, and platelets. Sulfatides interact with several cell adhesion molecules involved in hemostasis. Beta2-glycoprotein I is an anionic phospholipid-binding plasma protein, and the phospholipid-bound form is the target for most anti-phospholipid antibodies that are associated with recurrent thrombosis, miscarriages, and neurological symptoms. In this study, we examined whether beta2-glycoprotein I forms a complex with sulfatides and thereby becomes a target for anti-phospholipid antibodies. METHODS AND RESULTS: Beta2-glycoprotein I binds to surface-bound sulfatides but not to other glycolipids, such as ceramide, cerebrosides, sphingomyelin, or ganglioside. At a sulfatide coating density of 1 microg/well, beta2-glycoprotein I reaches half-maximal binding at 2.5 microg/mL, and the binding is saturated at 10 microg/mL. The binding of beta2-glycoprotein I also depends on the coating density of sulfatides in the well. At a constant beta2-glycoprotein I concentration of 5 microg/mL, maximal binding of beta2-glycoprotein I is observed at a coating density of 1 mug/well. The serum from 14 patients with anti-cardiolipin antibodies, a subset of anti-phospholipid antibodies, bound to sulfatide-bound beta2-glycoprotein I and previous absorption on cardiolipin-coated surfaces decreased the immunoreactivity toward sulfatide-beta2-glycoprotein I complex by >50% in 12 of 14 patients. Furthermore, immunoaffinity-purified anti-cardiolipin antibodies from 4 of 5 patients reacted with sulfatide-bound beta2-glycoprotein I. CONCLUSIONS: These results show that not only anionic phospholipids, as commonly known, but also sulfatides are targets for most anti-phospholipid antibodies. We therefore postulate that interactions of these antibodies with sulfatides may contribute to some of the clinical symptoms of the anti-phospholipid antibody syndrome.
Antibodies, Anticardiolipin↗
Sulfatides inhibit platelet adhesion to von Willebrand factor in flowing blood.
Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins such as von Willebrand factor (VWF), P-selectin, laminin and thrombospondin. Previous studies have localized the sulfatide-binding site of VWF to amino acid residues Gln626-Val646 in the A1 domain. The A1 domain also contains the binding site for platelet glycoprotein Ib (GP Ib), a site that has been reported to be distinct from the sulfatide-binding site. In this study, we analyzed the interaction of sulfatides with VWF and its effect on GP Ib-mediated platelet adhesion under flow conditions. Recombinant VWF A1 domain (rVWF-A1) bound specifically and saturably to sulfatides (half-maximal concentration of approximately 12.5 microg mL(-1)), binding that was blocked by dextran sulfate (IC(50) approximately equal to 100 microg mL(-1)) but not by heparin at concentrations up to 100 U mL(-1). Furthermore, sulfatides (125 microg mL(-1)) prevented the adhesion of platelets or glycocalicin-coupled polystyrene beads to a rVWF-A1-coated surface under high shear stress. In addition, plasma VWF prebound to a sulfatide-coated surface failed to support subsequent platelet adhesion. These results provide firm evidence that sulfatides bind the VWF A1 domain at a site overlapping the GP Ib-binding site.
Cloning, Molecular↗
The incidence of proximal deep vein thrombosis following total knee arthroplasty in an Asian population: a Doppler ultrasound study.
PURPOSE: To investigate the incidence of deep vein thrombosis (DVT) following total knee arthroplasty in an Asian population. METHODS: A prospective study of 149 consecutive cases of total knee arthroplasty done for osteoarthritis was conducted over a 5-year period. All patients underwent duplex ultrasonographic assessment of the lower limbs within the first postoperative week. RESULTS: The incidence of proximal DVT was found to be 4.38% in this study. Symptomology was statistically significant in predicting the presence of proximal DVT in all cases. General anaesthesia was associated with a statistically significant-higher incidence of DVT as compared with regional anaesthesia. There was a significant association between a sedentary lifestyle and the development of DVT. CONCLUSION: The incidence of proximal DVT in Asian patients after total knee arthroplasty is higher than that previously reported for this demographic group.
Adult↗
Role for sulfatides in platelet aggregation.
BACKGROUND: Sulfatides are sulfated glycosphingolipids present on the surface of oligodendrocytes, renal tubular cells, and certain tumor cells. They appear to be involved in nerve conduction and cell adhesion, but their precise physiological function is not known. METHODS AND RESULTS: Here, we show a novel role for sulfatides as a major ligand for P-selectin in platelet adhesion and aggregation. Sulfatides are expressed on the platelet surface, and platelets expressing sulfatides adhere to P-selectin. Both sulfatide micelles and sulfatide-binding recombinant malaria circumsporozoite protein (MCSP) inhibit this adhesion. In parallel, platelets and CHO cells expressing P-selectin adhere to sulfatides, and anti-P-selectin antibodies inhibit this adhesion. Furthermore, both anti-P-selectin antibodies and sulfatide antagonist MCSP significantly reverse platelet aggregation induced by ADP, collagen, or thrombin receptor-activating peptide, suggesting that sulfatide-P-selectin interactions are necessary for the formation of stable platelet aggregates. CONCLUSIONS: These results show that sulfatide interactions with P-selectin are important in platelet adhesion and platelet aggregation. The sulfatide interactions with P-selectin stabilize platelet aggregates, representing a new mechanism of platelet aggregation that may play a significant role in hemostasis and thrombosis.
Animals↗
Atherosclerosis, autoimmunity, and systemic lupus erythematosus.
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Animals↗
Cholesterol sulfate: a new adhesive molecule for platelets.
BACKGROUND: Cholesterol 3-sulfate is present on a variety of cells and in human LDL, and it has been found in atherosclerotic lesions of human aorta. Its precise biological role has not yet been described. METHODS AND RESULTS: In this study, we investigated the interaction of platelets with cholesterol sulfate. Platelets adhered in a concentration-dependent and saturable manner to cholesterol sulfate but did not adhere to cholesterol, cholesterol acetate, estrone sulfate, or dehydroepiandrosterone sulfate, suggesting that the specificity of this interaction is determined not only by the cholesterol moiety but also by the sulfate group. This adhesion did not increase after platelet activation, and it was not cation-dependent. Soluble cholesterol sulfate inhibited adhesion in a concentration-dependent manner. However, antibodies against glycoprotein Ib, glycoprotein IIb/IIIa, CD36, P-selectin, von Willebrand factor, or thrombospondin had no significant effect on platelet adhesion to cholesterol sulfate. Perfusion of whole blood in a parallel-plate flow chamber resulted in the rapid and progressive adhesion of platelets to cholesterol sulfate but not to cholesterol acetate or estrone sulfate. CONCLUSIONS: Cholesterol sulfate supports platelet adhesion and may be one of the factors determining the prothrombotic potential of atherosclerotic lesions.
Antibodies↗
Characterization of a novel autosomal dominant bleeding disorder in a large kindred from east Texas.
A large east Texas family with autosomal dominant inheritance of a novel bleeding disorder has been identified. The disorder is characterized clinically by easy bruising, life-threatening bleeding with trauma or surgery, and menorrhagia in affected women. Laboratory studies demonstrated prolongation of the prothrombin time and activated partial thromboplastin time in affected individuals. Paradoxically, assays of known coagulation factors are all within normal limits. To determine the molecular basis of this disease, a candidate gene linkage analysis in this kindred was done. Initially it was hypothesized that the cause of the disease in this family could be an antithrombin III (AT3) mutation that resulted in a constitutively active AT3 in the absence of heparin binding. Linkage studies using DNA from the family and an intragenic polymorphic marker within the AT3 gene showed that the disease mapped to this locus. The coding region and intron/exon junctions of AT3 were sequenced using the proband's DNA, but this analysis failed to identify a mutation. Additional family members were recruited for the study, and 16 polymorphic markers around the AT3 gene were analyzed. Using 2 recombinants, the critical interval for the defective gene was narrowed to approximately 1.5 Mb, centromeric to AT3. The factor V (FV) gene was mapped into the disease interval and sequenced; there were no mutations found. Elucidation of the genetic defect causing the bleeding disorder in this family may reveal a novel protein involved in the coagulation cascade.
Adult↗
Optimization of stacked half-hitch knots for arthroscopic surgery.
PURPOSE: This study was conducted to determine the optimal configuration of stacked half-hitch knots that would give the maximum knot-holding capacity (KHC). TYPE OF STUDY: Mechanical testing study. METHODS: The 2 types of suture materials tested were No. 1 PDS II monofilament and No. 2 Ethibond braided (Ethicon, Somerville, NJ) because these are the 2 most commonly used sutures in arthroscopic surgery. Twelve configurations of stacked half-hitch knots including 2 versions of the Revo knot were tested. Knots were tied between 2 steel hooks 7 mm apart on a manual knot-testing machine. The steel hooks were spread apart to break the knot and the KHC was determined by the peak load recorded on the digital force transducer. Maximum KHC was achieved for No. 1 PDS II sutures by locking the knot with 2 RHAPs (reversing half-hitch on alternate post) and, for No. 2 Ethibond sutures, by locking the knot with 3 RHAPs. Because of the possibility of PDS II sutures unraveling, it is recommended for both No. 1 PDS II and No. 2 Ethibond sutures, that all stacked half-hitches be locked with 3 RHAPs. By adding 1 RHAP to the Revo knot, failure by slippage can be eliminated. RESULTS: The recommended knots tied with braided No. 2 Ethibond sutures were 1.5 times stronger than knots tied with No. 1 PDS II sutures. Although there was variability in the KHC of the recommended knots tied by different surgeons using different knot tiers, the greatest difference in the mean KHC was less than 10%. CONCLUSIONS: Stacked half-hitch knots locked with 3 RHAPs are unlikely to fail by slippage.
Arthroscopy↗
A new role for P-selectin in shear-induced platelet aggregation.
BACKGROUND: P-selectin, expressed on platelets on activation, mediates rolling of platelets on endothelial cells, but its role in shear-induced platelet aggregation is not known. METHODS AND RESULTS: Platelets were exposed to either a single pulse (30 seconds) or 3 pulses (10 seconds) of high shear stress (150 to 200 dynes/cm(2)) each followed by low shear stress (10 dynes/cm(2)) for 4.5 minutes or 90 seconds, respectively, at 37 degrees C to resemble more closely in vivo conditions such as those in stenotic arteries. Under these conditions, platelet aggregation was significantly increased compared with low or high shear stress alone. Monoclonal anti-P-selectin antibodies inhibited shear-induced platelet aggregation, especially when induced by the combination of high and low shear stress, by approximately 70% and had an additive effect on the inhibition by abciximab (anti-glycoprotein (GP) IIb/IIIa antibody). However, anti-P-selectin antibody inhibited shear-induced platelet aggregation only at 37 degrees C, not at 22 degrees C, whereas abciximab inhibited shear-induced platelet aggregation at both 22 degrees C and 37 degrees C. This differential effect of anti-P-selectin antibody is explained by the finding that shear-induced P-selectin expression on platelets was observed mainly at 37 degrees C. CONCLUSIONS: These results indicate that pulsatile shear stress, which resembles flow conditions in stenotic arteries, induces significantly more platelet aggregation at 37 degrees C than monophasic shear stress. Under these conditions, we show a novel role for P-selectin in platelet aggregation distinct from that of GP IIb/IIIa, which may be of importance in the initiation of thrombosis associated with atherosclerotic lesions.
Analysis of Variance↗
P-selectin expression on platelets determines size and stability of platelet aggregates.
BACKGROUND: P-selectin mediates rolling of platelets and leukocytes on activated endothelial cells. After platelet activation, P-selectin is translocated from intracellular granules to the external membrane, whereas fibrinogen aggregates platelets by bridging glycoprotein (GP) IIb/IIIa between adjacent platelets. METHODS AND RESULTS: In this study, we define a novel role for P-selectin in platelet aggregation. Expression of P-selectin on the platelet surface correlated strongly with the mean platelet aggregate size. Inhibition of P-selectin binding to its ligand by either monoclonal anti-P-selectin antibodies directed against the lectin domain or soluble human P-selectin reversed platelet aggregation even when added up to 5 minutes after activation; however, fibrinogen binding to platelets was not affected. This deaggregating effect significantly reduced the maximal size and number of platelet aggregates. When added 1 minute after platelet activation, anti-P-selectin antibody achieved 95% to 100% of the deaggregating effect of EDTA, whereas the anti-GP IIb/IIIa antibody abciximab had no effect. Monoclonal antibodies against known P-selectin ligands, such as P-selectin GP ligand-1 (PSGL-1) or GP Ib, had no effect on platelet aggregation, suggesting a different ligand for P-selectin in platelet aggregate stabilization. In kinetic studies, P-selectin was maximally expressed 10 minutes after platelet activation, whereas maximal activation of GP IIb/IIIa occurred within the first 10 seconds, suggesting that P-selectin operates after fibrinogen binding to activated GP IIb/IIIa. CONCLUSIONS: These results indicate that P-selectin interaction with a ligand, different from PSGL-1 or GP Ib, stabilizes initial GP IIb/IIIa-fibrinogen interactions, allowing the formation of large stable platelet aggregates.
Abciximab↗
Case in point. Cerebral toxoplasmosis.
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AIDS-Related Opportunistic Infections↗
Monoclonal antibody light chain with prothrombinase activity.
Prothrombin is the precursor of thrombin, a central enzyme in coagulation. Autoantibodies to prothrombin are associated with thromboembolism, but the mechanisms by which the antibodies modulate the coagulation processes are not understood. We screened a panel of 34 monoclonal antibody light chains isolated from patients with multiple myeloma for prothrombinase activity by an electrophoresis method. Two light chains with the activity were identified, and one of the light chains was characterized further. The prothrombinase activity eluted from a gel-filtration column run in denaturing solvent (6 M guanidine hydrochloride) at the characteristic positions of the light chain dimer and monomer. A constant level of catalytic activity was observed across the width of the light chain monomer peak, assessed as the cleavage of IEGR-methylcoumarinamide, a peptide substrate corresponding to residues 268-271 of prothrombin. Hydrolysis of this peptide by the light chain was saturable and consistent with Michaelis-Menten-Henri kinetics (K(m) 103 microM; k(cat) of 2.62 x 10(-)(2)/min). Four cleavage sites in prothrombin were identified by N-terminal sequencing of the fragments: Arg(155)-Ser(156), Arg(271)-Thr(272), Arg(284)-Thr(285), and Arg(393)-Ser(394). The light chain did not cleave radiolabeled albumin, thyroglobulin, and annexin V under conditions that readily permitted detectable prothrombin cleavage. Two prothrombin fragments (M(r) 55 000 and 38 000), were isolated by anion-exchange chromatography and were observed to cleave a thrombin substrate, tosyl-GPR-nitroanilide. Conversion of fibrinogen to fibrin was accelerated by the prothrombin fragments generated by the light chain. These finding suggest a novel mechanism whereby antibodies can induce a procoagulant state, i.e., prothrombin activation via cleavage of the molecule.
Antibodies, Monoclonal↗
Primary antiphospholipid antibody syndrome with mutations in the phospholipid binding domain of beta(2)-glycoprotein I.
beta(2)-Glycoprotein I, an anionic phospholipid-binding 50-kDa plasma protein, circulates in the plasma at a concentration of 30-200 microg/ml. Its physiological role remains uncertain, but an important clue to this role is suggested by the finding that antibodies to this protein are frequently found in patients with antiphospholipid antibodies and thrombosis. beta(2)-Glycoprotein I belongs to the complement control protein (CCP) superfamily with five CCP domains. The fifth CCP domain of beta(2)-glycoprotein I has a unique structure and contains a stretch of positively charged amino acids that mediates the binding to phospholipids. This interaction may mediate the clearance of anionic phospholipid-containing surfaces from the circulation. Mutations in this domain affect its binding to phospholipids. We have identified a patient with primary antiphospholipid syndrome who is a compound heterozygous for two mutations in the fifth CCP. One mutation is located in exon 7 (codon 306), and the second mutation is in exon 8 (codon 316). The mutant beta(2)-glycoprotein I was present in normal quantities in his plasma but did not bind to cardiolipin. He had recurrent deep vein thrombosis and pulmonary embolism at age 28 and a thrombotic stroke at age 35, with no other identifiable risk factor for a hypercoagulable state. This report offers some insight into the mechanism of formation of antiphospholipid antibodies and suggests the possible role of the deficiency of beta(2)-glycoprotein I in the pathogenesis of thrombosis.
Adult↗
Thrombin receptor activating peptide (SFLLRN) potentiates shear-induced platelet microvesiculation.
Shear-induced activation of platelets plays a major role in vascular thrombosis. Shear stress tends to increase both platelet aggregation and procoagulant activity. One mechanism for increased procoagulant activity is promotion of the transbilayer movement of anionic phospholipids from the inner to the outer leaflet of the platelet membrane bilayer. This is accompanied by vesiculation of the platelet membrane, resulting in the formation of procoagulant membrane particles called microvesicles. In this study we have examined the effect of various platelet agonists on shear-induced platelet microvesiculation and the development of platelet procoagulant activity. Normal citrated whole blood was subjected to laminar shear rate up to 12,500 sec(-1) (shear stress approximately 375 dyne/cm2) in a cone-and-plate viscometer, and the formation of platelet microvesicles was measured by flow cytometry under different conditions. Elevated levels of shear stress induced significant microvesiculation. We investigated the effects of adenosine diphosphate, epinephrine, thromboxane A2 analog, collagen, and thrombin receptor activation peptide (SFLLRN) on shear-induced platelet microvesiculation. The thrombin peptide significantly increased shear-induced microvesicle formation. In contrast, under similar conditions, the other agonists had no significant effect on shear-induced microvesiculation. These studies suggest that thrombin formed in the vicinity of primary hemostatic plugs in areas of elevated shear stress may have a major role in the propagation of thrombi by potentiating shear-induced platelet microvesiculation.
15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
Biomechanical study on the effect of twisted human patellar tendon.
OBJECTIVE: To compare the stiffness and maximum strength between the untwisted and twisted free-tendon. DESIGN: 22 twisted and untwisted sectioned-specimens of human cadaver patellar tendons were used and pulled to failure to obtain load-deformation profiles from which stiffness, maximum load to failure and elastic elongation limit were derived. BACKGROUND: In the reconstruction of the deficient anterior cruciate ligament, the use of the central one-third of the patellar tendon is a well-established procedure in which, prior to insertion, the tendon graft may be twisted to mimic the natural orientation of the anterior cruciate ligament in the knee joint. RESULTS: The untwisted tendons had a mean stiffness of 36.5 kg/mm (SD, 16.6 kg/mm) and maximum load of 165.9 kg (SD, 86.8 kg). With a 90 degrees twist, the average stiffness of the twisted tendon was 66.5 kg/mm (SD, 25.4 kg/mm), with maximum load at 364.5 kg (SD, 109.9 kg), an increase of over 100%. The elastic elongation limit, or allowable elongation before permanent deformation or failure, was significantly larger in twisted tendons by 35%. CONCLUSION: Twisting increased the resistance to deformation of the tendon in this study. Relevance The finding supports the surgical practice of pre-twisting tendon grafts for anterior cruciate ligament reconstruction, based on the premise that a stronger and stiffer graft provides a more favourable outcome.
Adult↗
Prothrombin cleaving antibody light chains.
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Amino Acid Sequence↗