Tissue factor (TF)-induced coagulation was compared in contact pathway suppressed human blood from normal, factor VIII-deficient, and factor XI-deficient donors. The progress of the reaction was analyzed in quenched samples by immunoassay and immunoblotting for fibrinopeptide A (FPA), thrombin-antithrombin (TAT), factor V activation, and osteonectin. In hemophilia A blood (factor VIII:C <1%) treated with 25 pmol/L TF, clotting was significantly delayed versus normal, whereas replacement with recombinant factor VIII (1 U/mL) restored the clot time near normal values. Fibrinopeptide A release was slower over the course of the experiment than in normal blood or hemophilic blood with factor VIII replaced, but significant release was observed by the end of the experiment. Factor V activation was significantly impaired, with both the heavy and light chains presenting more slowly than in the normal or replacement cases. Differences in platelet activation (osteonectin release) between normal and factor VIII-deficient blood were small, with the midpoint of the profiles observed within 1 minute of each other. Thrombin generation during the propagation phase (subsequent to clotting) was greatly impaired in factor VIII deficiency, being depressed to less than 1/29 (<1.9 nmol TAT/L/min) the rate in normal blood (55 nmol TAT/L/min). Replacement with recombinant factor VIII normalized the rate of TAT generation. Thus, coagulation in hemophilia A blood at 25 pmol/L TF is impaired, with significantly slower thrombin generation than normal during the propagation phase; this reduced thrombin appears to affect FPA production and factor V activation more profoundly than platelet activation. At the same level of TF in factor XI-deficient blood (XI:C <2%), only minor differences in clotting or product formation (FPA, osteonectin, and factor Va) were observed. Using reduced levels of initiator (5 pmol/L TF), the reaction was more strongly influenced by factor XI deficiency. Clot formation was delayed from 11.1 to 15.7 minutes, which shortened to 9.7 minutes with factor XI replacement. The maximum thrombin generation rate observed (∼37 nmol TAT/L/min) was approximately one third that for normal (110 nmol/L TAT/min) or with factor XI replacement (119 nmol TAT/L/min). FPA release, factor V activation, and release of platelet osteonectin were slower in factor XI-deficient blood than in normal blood. The data demonstrate that factor XI deficiency results in significantly delayed clot formation only at sufficiently low TF concentrations. However, even at these low TF concentrations, significant thrombin is generated in the propagation phase after formation of the initial clot in hemophilia C blood.

Blood clotting involves a multitude of proteins that act in concert in response to vascular injury to produce the procoagulant enzyme alpha-thrombin, which in turn is responsible for the generation of the fibrin plug. However, while generation of the fibrin plug is required for the arrest of excessive bleeding, unregulated clotting will result in the occlusion of the blood vessels and thrombosis. Thus, the regulation of the delicate balance between the procoagulant and anticoagulant mechanisms is of extreme importance for survival. While the majority of proteins involved in blood coagulation circulate as inactive zymogens that require proteolytic activation in order to function, approximately 1% of the circulating factor VII molecules are active. Factor VIIa, possess a serine protease active site, has poor catalytic activity, and is not inhibited by the circulating stoichiometric protease inhibitors. Following injury to the vasculature and subsequent exposure of the integral membrane glycoprotein, tissue factor (TF), the circulating factor VIIa molecules can bind to the exposed TF forming the extrinsic tenase complex (TF/factor VIIa) and initiate the blood coagulation process. Formation of the TF/factor VIIa complex increases the catalytic efficiency of the enzyme by four orders of magnitude when compared with factor VIIa alone. This cell-associated enzymatic complex initiates a series of enzymatic reactions, leading to the generation of alpha-thrombin and ultimately to the formation of the fibrin plug. The procoagulant enzymatic complexes (i.e., prothrombinase, intrinsic tenase, and extrinsic tenase) are similar in structure and composed of an enzyme, a cofactor, and the substrate associated on a cell surface in the presence of divalent metal ions. While the activity of the extrinsic tenase complex is limited by the availability (exposure) of its cell-associated cofactor (TF) it is remarkable that the activities of both the prothrombinase complex (factor Va/factor Xa) as well as the intrinsic tenase complex (factor VIIIa/factor IXa) are limited by the presence of the two soluble, nonenzymatic cofactors, factor Va and factor VIIIa. Factor Va and factor VIIIa, which are very similar in structure and function, are required for prothrombinase and intrinsic tenase activities, respectively, because both cofactors express a dual function in their respective complexes, acting as an enzyme receptor and catalytic effector on the cell surface. The cofactors derive from inactive plasma precursors by regulatory proteolytic events that involve alpha-thrombin. In general, bleeding tendencies are usually associated with defects in the activation of one of the zymogens or the cofactors of the procoagulant complexes. However, the activity of all of the complexes is also limited by the availability of an adequate membrane surface provided by endothelial cells, platelets, and monocytes. The cell surface provides a site for the recruitment of the appropriate proteins and allows for fast and efficient clot formation. In the absence o...

SummaryThe influence of platelets on tissue factor (TF)-initiated thrombin generation in a reconstituted model of blood coagulation and in whole blood was evaluated. No thrombin generation was observed over 15 min in the reconstituted model when either TF or platelets and phospholipids were omitted. At 25 pM TF, the rates of thrombin generation were platelet and PCPS concentration-dependent and achieved maximum (1.0 nM/s) in the physiological range of platelet concentration. Similar rates were achieved in the absence of platelets when 1-2 μM phospholipid was used. However, the maximum rates of thrombin generation (5.2-6.0 nM/s) and the shortest initiation phase (1 min) were attained between 25 and 100 μM phospholipid. In the reconstituted model, an increase in platelet concentration from 0.125 × 108/ml to 0.5 × 108/ml decreased the duration of the initiation phase (in the absence of phospholipids) from 4.3 min to 2 min. Further increases in platelet concentration did not affect this phase. Sequential whole blood studies were conducted in blood of a chemotherapy patient who developed reduced platelet counts. The TF (12.5 pM) initiated clotting of patient’s blood was accelerated from ~10 min to 5 min when the platelet concentration increased from 0.05 × 108/ml to 0.11 × 108/ml. Clotting times were essentially unchanged for platelet concentrations exceeding 0.5 × 108/ml (range 0.5-3.1 × 108/ml). Similarly, clotting of whole blood obtained from healthy volunteers was not affected by the platelet count, which varied from 1.5 × 108/ml to 3.1 × 108/ml (4.0 ± 0.5 min). The data obtained in both models are consistent with in vivo observations that clinical bleeding is most likely to occur at platelet counts <0.1 × 108/ml.Portions of this work were presented at the 40th Annual Meeting of the American Society of Hematology, December 4-8, 1998, Miami Beach, Florida (abstracts #140 and #738).

IntroductionThe importance of the platelet in normal hemostasis and in disease states, such as atherosclerosis and thrombosis, is well established. [1][2][3][4][5] A particularly germane example is the platelet-rich thrombus, which forms on fissured or ruptured atherosclerotic plaques. [3][4][5] Although the slow formation of the underlying plaques is a progressive disease that serves to narrow the lumen of the vessel over an extended period of time, it is the sudden and catastrophic superposition of the platelet-rich thrombotic occlusion on these lesions that is central to nearly all ischemic coronary events. General acceptance that the ultimate occlusive event is thrombotic in nature has led to the development of strategies that attempt to ameliorate, inhibit, or reverse this final phase of the coronary disease process. Some of the newest strategies focus on inhibition of platelet functions, which contribute to the formation of the thrombotic occlusion.The generation of thrombin during the blood clotting process can be divided into 2 semidiscrete intervals, the initiation and propagation phases. 6 During the initiation phase nanomolar amounts of thrombin and picomolar amounts of other coagulation enzymes are generated. Clot occurs at the inception of the propagation phase, which is characterized by rapid and near quantitative thrombin generation. By this point activation of platelets and cleavage of factor V and factor VIII are almost complete. 7 In normal blood, the thrombin activation process is independent of platelet or factor V activation, but is limited by the generation of factor Xa. 6,7 Normal platelet function has been divided into 3 processes: adhesion, aggregation, and activation (including surface phospholipid rearrangements and secretory pathways [8][9][10][11] ). These functions bestow on the platelet its antihemorrhagic qualities and are regulated by a variety of mechanisms. 12 The most potent platelet agonist is ␣-thrombin, the central enzyme of coagulation that controls hemorrhage in vivo. Thrombin is generated from the inactive circulating precursor prothrombin via the tissue factor (TF) pathway of coagulation [13][14][15][16] ; this serine protease activates platelets via specific cell surface receptors, leading to secretion, aggregation, changes in platelet morphology, and expression of a procoagulant, phospholipid-equivalent, surface. 9-11 Aggregated thrombin-activated platelets form the basis of the thrombus in normal hemostasis and provide the surface on which the complexdependent reactions of the TF pathway are localized. Furthermore, thrombin amplifies its own generation by activating circulating plasma procofactors (factor V and factor VIII), critical components of the pathway's proteolytic complex catalysts, which also serve to localize proteolytic activity to the surface of the activated platelet. 6,17 Thrombin converts fibrinogen by limited proteolysis to fibrin, which polymerizes throughout the growing thrombus rendering added stability. [18][19][20] The importance of TF-induced t...

The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N.A2C peptides, with intermediate activity in each species. Quantitation of the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307-713 (A2N.A2C + A2C-peptide), leaving behind the membrane-bound A1.LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with [APC], reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (Kd = 7 nM), suggesting that the membrane-bound LC.A1 product would act to sequester APC. This inhibitory interaction (LC.A1.APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.