Contribution of Platelet-derived Factor Va to Thrombin Generation on Immobilized Collagen- and Fibrinogen-adherent Platelets (original) (raw)
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Journal of Biological Chemistry, 2001
Recent studies have indicated that factor Va bound to activated platelets is partially protected from inactivation by activated protein C (APC). To explore whether this sustained factor Va activity could maintain ongoing thrombin generation, the kinetics of platelet factor Vadependent prothrombinase activity and its inhibition by APC were studied. In an attempt to mimic physiologically relevant conditions, platelets were adhered to collagen type I-coated discs. These discs were then spun in solutions containing prothrombin and factor Xa either in the absence or presence of APC. The experiments were performed in the absence of platelet-derived microparticles, with thrombin generation and inhibition confined to the surface of the adherent platelets. APC completely inactivated platelet-associated prothrombinase activity with an overall second order rate constant of 3.3 ؋ 10 6 M ؊1 s ؊1 , which was independent of the prothrombin concentration over a wide range around the apparent K m for prothrombin. Kinetic studies on prothrombinase assembled at a planar phospholipid membrane composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine revealed a similar second order rate constant of inhibition (2.5 ؋ 10 6 M ؊1 s ؊1). Collectively, these data demonstrate that ongoing platelet factor Va-dependent thrombin generation at the surface of collagen-adherent platelets is effectively inhibited by APC. No differences were observed between the kinetics of APC inactivation of plasma-derived factor Va or platelet factor Va as part of the prothrombinase associated with, respectively, a planar membrane of synthetic phospholipids or collagen-adherent platelets.
Journal of Clinical Investigation, 1985
Platelet adhesion to monomeric collagen types I and III, which were purified from human umbilical arteries, was studied in a perfusion chamber under well defined flow conditions. For this purpose, glass coverslips were coated with 20-30 'sg/cm2 of collagen types I and III by spraying a solution of these collagens with a retouching air brush. Platelet deposition increased with the time of perfusion. Adhesion to both collagen types was similar in the first 3 min, but increased platelet deposition occurred on collagen type III after 3 min due to thrombus formation. Adhesion at a shear rate of 800 s-' was strongly impaired with plasma of a patient with von Willebrand's disease or with fibronectin-free plasma. Addition of purified fibronectin to fibronectin-free plasma restored adhesion to the level obtained with normal plasma. Platelet deposition in normal plasma increased with increasing shear rates. Platelet deposition in VWD-plasma was normal at 490 s-', but there was no increase at higher shear rates. Platelet deposition in fibronectin-free plasma was diminished at all shear rates studied from 490 to 1,300 sO.
2000
Factor V (FV) present in platelet ␣-granules has a significant but incompletely understood role in hemostasis. This report demonstrates that a fraction of platelets express very high levels of surface-bound, ␣-granule FV on simultaneous activation with 2 agonists, thrombin and convulxin, an activator of the collagen receptor glycoprotein VI. This subpopulation of activated platelets represents 30.7% ؎ 4.7% of the total population and is referred to as convulxin and thrombin-induced-FV (COAT-FV) platelets. COAT-FV platelets are also observed on activation with thrombin plus collagen types I, V, or VI, but not with type III. No single agonist examined was able to produce COAT-FV platelets, although ionophore A23187 in conjunction with either thrombin or convulxin did generate this population. COAT-FV platelets bound annexin-V, indicating exposure of aminophospholipids and were enriched in young platelets as identified by the binding of thiazole orange. The functional significance of COAT-FV platelets was investigated by demonstrating that factor Xa preferentially bound to COAT-FV platelets, that COAT-FV platelets had more FV activity than either thrombin or A23187activated platelets, and that COAT-FV platelets were capable of generating more prothrombinase activity than any other physiologic agonist examined. Microparticle production by dual stimulation with thrombin and convulxin was less than that observed with A23187, indicating that microparticles were not responsible for all the activities observed. These data demonstrate a new procoagulant component produced from dual stimulation of platelets with thrombin and collagen. COAT-FV platelets may explain the unique role of ␣-granule FV and the hemostatic effectiveness of young platelets. (Blood. 2000;95:1694-1702)
Biochemical and Biophysical Research Communications, 2001
The extravascular localization of tissue factor (TF), the central initiator of coagulation, is thought to ensure that thrombus formation is prevented in the intact vessel. We observed that during a 5-min stimulation of human blood with collagen (type I), TF antigen appeared on the surface of platelets adhering to leukocytes. The rapidly presented intravascular TF was competent to start the coagulation cascade. The isolated platelets from healthy donors contained appreciable amounts of the TF protein, while no TF antigen was detected in the neutrophils and rapidly isolated monocytes. Direct interactions with the neutrophils and monocytes were apparently necessary to activate the platelet-associated TF. This was most likely mediated by inactivation of tissue factor pathway inhibitor through leukocyte elastase. In summary, the leukocyte-elicited activation of the platelet TF participates in the rapid initiation of coagulation by collagen.
Platelets and Thrombin Generation
Arteriosclerosis, Thrombosis, and Vascular Biology, 2002
This review examines the evidence that platelets play a major role in localizing and controlling the burst of thrombin generation leading to fibrin clot formation. From the first functional description of platelets, it has been recognized that platelets supply factors that support the activation of prothrombin. Studies have demonstrated that on activation, the amount of one specific lipid, phosphatidylserine, is significantly increased on the outer leaflet of platelet membranes. When it was found that phosphatidylserine containing lipid extracts could be substituted for platelets in clotting assays, this suggested the possibility that changes in platelet lipid composition were necessary and sufficient to account for platelet surface thrombin generation. Because a growing body of data suggest that platelet-binding proteins provide much of the specificity for platelet thrombin generation, we review in this report data suggesting that changes in lipid composition are necessary but not sufficient to account for platelet surface regulation of thrombin generation. Also, we review data suggesting that platelets from different individuals differ in their capacity to generate thrombin, whereas platelets from a single subject support thrombin generation in a reproducible manner. Individual differences in platelet thrombin generation might be accounted for by differences in platelet-binding proteins. (Arterioscler Thromb Vasc Biol. 2002;22:1381-1389.)
Biomaterials, 2000
Procoagulant (activated) platelets provide a site for assembly of the prothrombinase complex which can rapidly convert prothrombin into thrombin (a potent inducer of clot formation). Previously, we reported that adhesion of platelets to surfaces preadsorbed with blood plasma caused them to become procoagulant. In the present study we investigated the e!ect of adsorbed adhesion proteins ("brinogen (Fg), "bronectin (Fn), von Willebrand factor (vWF) and vitronectin (Vn)) on the procoagulant activity of adherent platelets. Adsorbed Fn, vWF and Fg promoted platelet adhesion in the following order: Fn(vWF"Fg. However, these proteins promoted platelet activation (thrombin generation per adherent platelet) in the following order: Fg(Fn(vWF. Adsorption with a series of dilutions of normal plasma, serum, and plasmas de"cient in or depleted of von Willebrand factor (de-vWF), "bronectin (de-Fn), vitronectin (de-Vn), or both vitronectin and "bronectin (de-VnFn) resulted in varied platelet adhesion, but little di!erence in platelet activation. However, preadsorption with dilute de-vWF plasma induced lower procoagulant activity than normal plasma. Preadsorption with normal plasma resulted in higher levels of platelet activation than preadsorption with Fg, suggesting that adsorption of plasma proteins other than Fg caused the high levels of activation observed for plasma preadsorbed surfaces.