Human platelets and factor XI. Localization in platelet membranes of factor XI-like activity and its functional distinction from plasma factor XI.

Because human platelets participate in the contact phase of intrinsic coagulation and contain a Factor XI-like coagulant activity, the nature of the Factor XI-like activity was examined and compared with purified plasma Factor XI. The platelet factor XI-like activity was sedimented with the particulate fraction of a platelet lysate, was inactivated by heat (t(1/2) 3.5 min, 56 degrees C), was not a nonspecific phospholipid activity, and was destroyed by treatment with Triton X-100. Isolated platelet membranes were four-fold enriched in Factor XI activity and similarly enriched in plasma membrane marker enzymes. The Factor XI-like activity of platelet membranes was detected only when assayed in the presence of kaolin, which suggests that it is present in an unactivated form and can participate in contact activation. Concanavalin A inhibited the Factor XI-like activity of platelet lysates and platelet membranes but not of plasma or purified Factor XI. A platelet membrane-Factor XI complex was isolated after incubation of membranes with purified Factor XI. The Factor XI activity of the platelet membrane-plasma Factor XI complex was inhibited by concanavalin A, whereas unbound plasma Factor XI retained activity. An antibody raised against plasma Factor XI inhibited the in vitro Factor XI activity of plasma and of the platelet membrane-plasma Factor XI complex but had no effect on the endogenous Factor XI-like activity of washed lysed platelets or isolated platelet membranes. Washed platelets and isolated platelet membranes obtained from a Factor XI-deficient donor without a history of excessive bleeding had normal quantities of platelet Factor XI-like activity and normal behavior in the contact phase of coagulation (collagen-induced coagulant activity). These results indicate that platelet membranes contain an endogenous Factor XI-like activity that is functionally distinct from plasma Factor XI.

Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug.

Platelet receptor-mediated factor X activation by factor IXa. High-affinity factor IXa receptors induced by factor VIII are deficient on platelets in Scott syndrome.

We have studied factor IXa binding and factor X activation with normal platelets and with platelets obtained from a patient with a bleeding disorder and an isolated deficiency of platelet procoagulant activity termed Scott syndrome. In the absence of factor VIIIa and factor X, normal, thrombin-treated platelets exposed 560 +/- 35 sites for factor IXa with a Kd of 2.75 +/- 0.27 mM, compared with 461 +/- 60 sites per patient platelet with Kd of 3.2 +/- 0.33 nM. The addition of factor VIIIa and factor X resulted in a decrease in the Kd for normal platelets to 0.68 nM but had no effect on the Kd for patient platelets. The concentrations of factor IXa required for half-maximal rates of factor X activation for normal (0.52 nM) and patient platelets (2.5 nM) were similar to those determined from equilibrium binding studies. Kinetic parameters for factor X activation by factor IXa showed that the Km and Kcat were identical for normal and patient platelets in the absence of factor VIIIa. In the presence of factor VIIIa, and kcat for patient platelets (163 min-1) was only 33% of that for normal platelets (491 min-1): This result can be explained by the difference in affinity for factor IXa between normal and patient platelets in the presence of factor VIIIa, suggesting impaired factor VIIIa binding to Scott syndrome platelets.

Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX.

The kinetics of activation of human Factor IX by human Factor XIa was studied by measuring the release of a trichloroacetic acid-soluble tritium-labeled activation peptide from Factor IX by a modification of a method described for bovine Factor IX activation by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707). Initial rates of trichloroacetic acid-soluble 3H-release were linear over 10-30 min of incubation of Factor IX (88 nM) with CaCl2 (5 mM) and with pure (greater than 98%) Factor XIa (0.06-1.3 nM), which was prepared by incubating human Factor XI with bovine Factor XIIa. Release of 3H preceded the appearance of Factor IXa activity, and the percentage of 3H released remained constant when the mole fraction of 3H-labeled and unlabeled Factor IX was varied and the total Factor IX concentration remained constant. A linear correlation (r greater than 0.98, P less than 0.001) was observed between initial rates of 3H-release and the concentration of Factor XIa, measured by chromogenic assay and by radioimmunoassay and added at a Factor IX:Factor XIa molar ratio of 70-5,600. Kinetic parameters, determined by Lineweaver-Burk analysis, include Km (0.49 microM) of about five- to sixfold higher than the plasma Factor IX concentration, which could therefore regulate the reaction. The catalytic constant (kcat) (7.7/s) is approximately 20-50 times higher than that reported by Zur and Nemerson (Zur, M., and Y. Nemerson, 1980, J. Biol. Chem., 255:5703-5707) for Factor IX activation by Factor VIIa plus tissue factor. Therefore, depending on the relative amounts of Factor XIa and Factor VIIa generated in vivo and other factors which may influence reaction rates, these kinetic parameters provide part of the information required for assessing the relative contributions of the intrinsic and extrinsic pathways to Factor IX activation, and suggest that the Factor XIa catalyzed reaction is physiologically significant.

Blood coagulation factor XIa binds specifically to a site on activated human platelets distinct from that for factor XI.

Binding of 125I-Factor XIa to platelets required the presence of high molecular weight kininogen, was enhanced when platelets were stimulated with thrombin, and reached a plateau after 4-6 min of incubation at 37 degrees C. Factor XIa binding was specific: 50- to 100-fold molar excesses of unlabeled Factor XIa prevented binding, whereas Factor XI, prekallikrein, Factor XIIa, and prothrombin did not. When washed erythrocytes, added at concentrations calculated to provide an equivalent surface area to platelets, were incubated with Factor XIa, only a low level of nonspecific, nonsaturable binding was detected. Factor XIa binding to platelets was partially reversible and was saturable at concentrations of added Factor XIa of 0.2-0.4 microgram/ml (1.25-2.5 microM). The number of Factor XIa binding sites on activated platelets was estimated to be 225 per platelet (range, 110-450). We conclude that specific, high affinity, saturable binding sites for Factor XIa are present on activated platelets, are distinct from those previously demonstrated for Factor XI, and require the presence of high molecular weight kininogen.

Mechanism of activation of coagulation factor XI by factor XIIa studied with monoclonal antibodies.

The interaction of Factor XIIa with Factor XI was investigated using two monoclonal antibodies, one (3Cl) directed against the heavy chain of Factor XIa and the other (5F4) against its light chain. 3C1 either as intact IgG or as Fab' fragment, enhanced the rate of Factor XIa generation in the fluid phase but inhibited it in the presence of kaolin and high molecular weight (HMW) kininogen. In contrast, the Fab' fragments of 5F4 inhibited only the fluid phase activation and had no effect on the surface-mediated activation. 3C1 was found to block the binding of Factor XI to HMW kininogen, whereas 5F4 did not. We conclude: a domain on the heavy chain region of Factor XI is essential for binding to HMW kininogen and for optimal surface-mediated activation by Factor XIIa; and binding of 3C1 to Factor XI changes its conformation rendering it a more favorable substrate for Factor XIIa in the fluid phase.

Active release of human platelet factor VIII-related antigen by adenosine diphosphate, collagen, and thrombin.

Platelet Factor VIII-related antigen (VIIIR:Ag) represents a significant proportion of the total circulating VIIIR:Ag pool. However, its participation in the events of primary hemostasis has not been shown. We now report that platelet-contained VIIIR:Ag is released from platelets by collagen, ADP and thrombin. The concentrations of these agonists, required for VIIIR:Ag release, are the same or lower than those required for release of serotonin, lysosomal enzymes, or fibrinogen. This release has the features of an energy-dependent secretory response because it is blocked by the metabolic inhibitors, antimycin A and 2-deoxy-D-glucose. The electrophoretic characteristics of the VIIIR:Ag released by collagen and ADP are similar to those of plasma VIIIR:Ag. However, thrombin-released platelet VIIIR:Ag differs from that of plasma in that the less anodal forms are relatively depleted. These differences do not appear to be the result of proteolytic degradation of platelet-derived VIIIR:Ag, but may reflect interactions between specific molecular forms of VIIIR:Ag and the platelet membrane. These studies suggest mechanisms by which platelet-contained VIIIR:Ag may contribute to the primary events of hemostasis.

Template bleeding time and clinical hemorrhage in myeloproliferative disease.

In 32 patients with myeloproliferative disorders (MPD), correlations were made among clinical observations of hemorrhagic tendency, template Ivy bleeding time, and platelet aggregation studies. Bleeding time was commonly prolonged, particularly in myelofibrosis. In two cases, this prolongation appeared to reflect a defect in platelet function, which resulted in clinical bleeding. Prolongation of bleeding time did not correlate with degree of thrombocytosis. Two patients with thrombocytosis had serious clinical bleeding at a time when bleeding time was normal. Of the patients, 35% had abnormal findings from aggregation studies, but there was no correlation between aggregation studies and prolongation of bleeding time or clinical hemorrhage. We conclude that bleeding in MPD arises either from a defect in platelet function, which is reflected in a prolonged bleeding time, or from thrombocytosis.

Platelet membrane-mediated coagulation protease complex assembly.

Platelet membranes provide procoagulant surfaces for the assembly and expression of a variety of coagulation protease complexes. These assembled complexes promote the proteolytic activation of various coagulation proteins resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable receptors for factor (F) XI, FXIa, FIX, FIXa, FX, FXa, FV, FVa, prothrombin, and thrombin. The molecular mechanisms involved in the assembly of the intrinsic tenase and prothrombinase enzyme-cofactor complexes on platelet membranes are the subject of intense investigation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant cofactors and proteases, is currently unresolved. In this article, we review some of these platelet receptor-mediated coagulation protein interactions and discuss platelet receptor-mediated F-X activation as a point of attack for the development of antithrombotic agents.

The assembly of the factor X-activating complex on activated human platelets.

Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant factors and proteases, is currently unresolved. The interaction between coagulation proteins and platelets is critical to the maintenance of normal hemostasis and is pathogenetically important in human disease.