Platelet Receptor Occupancy with Factor IXa Promotes Factor X Activation

Ahmad, Shafeeque; Rawala-Sheikh, Razia; Walsh, Peter N.

doi:10.1016/s0021-9258(19)47211-6

Cited by 59 publications

(43 citation statements)

References 23 publications

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“…The K'A and Vsat values obtained in reciprocal titrations of factors IXa and VIIIa were equivalent within experimental error. These results are consistent with hypothetical assembly mechanisms proposed by other investigators (10), and suggest a 1:1 molecular ratio for the functional interaction between factors IXa and VIII on the platelet membrane.…”

Section: Discussionsupporting

confidence: 93%

“…In functional titrations, concentrations of factor IXa required to achieve halfmaximal rates of factor X activation on platelets were found to be 0.5 nM (8). The value of this functional parameter is close to the Ka determined in equilibrium binding experiments (10). The corresponding functional parameter for factor VIII has not been determined.…”

mentioning

confidence: 52%

“…Kinetic and functional characteristics of monocyte-mediated reactions of the extrinsic and common coagulation pathways have been examined in detail before (1)(2)(3)(4)(5)(6)(7). In contrast, little is catalyzed by a membrane-associated enzymatic complex formed by a protease component, factor IXa, and an essential cofactor, factor VIIIa (8)(9)(10). This cofactor is synthesized as a single polypeptide with 2351 residues, but it is recovered from plasma as a heterogeneous mixture of polypeptides with molecular weights ranging from 80 to 210 kD (11,12).…”

mentioning

confidence: 99%

“…Equilibrium binding studies of factor VIII interaction with platelet membranes and with artificial lipid vesicles have yielded a dissociation constant (Ka) of 2-4 nM (13,14). It is also known that interaction between factors VIII and IXa on a lipid membrane is indispensable for expression of biologically significant coagulant activity (8)(9)(10). In functional titrations, concentrations of factor IXa required to achieve halfmaximal rates of factor X activation on platelets were found to be 0.5 nM (8).…”

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confidence: 99%

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Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

McGee

Hensler

1992

The Journal of Experimental Medicine

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SummaryGeneration of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombinactivated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaC12, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The KIA values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/u and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane. ,• ctivation of the coagulation cascade is mediated by membrane-dependent protease complexes historically grouped into three different pathways: extrinsic, intrinsic, and common. The monocyte/macrophage, a central element in the inflammatory response, has been shown to provide appropriate membrane sites for efficient assembly of coagulation proteases. Kinetic and functional characteristics of monocyte-mediated reactions of the extrinsic and common coagulation pathways have been examined in detail before (1-7). In contrast, little Parts of this study have been published in abstract form. . FASEB (Fed. Am. Soc ExI~ Biol.) J. 4:2278a information is available on kinetic characteristics of intrinsic pathway rea...

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Section: Discussionsupporting

confidence: 93%

mentioning

confidence: 52%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

McGee

Hensler

1992

The Journal of Experimental Medicine

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“…FXa Generation Assay. FXa generation was assayed by a modification of our previously published method (Ahmad et al, 1989b;Rawala-Sheikh et al, 1990). All proteins were diluted in 50 mM HEPPS, 175 mM NaCl, pH 8.1 (buffer B).…”

Section: Methodsmentioning

confidence: 99%

Annexin V Inhibition of Factor IXa-Catalyzed Factor X Activation on Human Platelets and on Negatively-Charged Phospholipid Vesicles

1996

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Annexin V was found to inhibit factor IXa-catalyzed factor X activation on both thrombin-activated human platelets and artificial lipid vesicles containing phosphatidylserine, supporting previous observations of the importance of negatively-charged lipid in potentiating the reaction. Annexin V reduced the Vmax of factor X activation in factor IXa titrations on the platelet surface with an IC50 of 4 nM in the absence of thrombin-activated factor VIII (factor VIIIa), and 4.5 nM in its presence, whereas there was no effect on the EC50,FIXa. This noncompetitive inhibition is consistent with interference of recognition of the factor IXa binding site on the platelet, which was confirmed by equilibrium binding of [125I]-factor IXa to thrombin-activated platelets where, in the absence of factor VIIIa and factor X, annexin V reduced the number of factor IXa binding sites/platelet from 610 to 320, without changing the Kd,app. In the presence of factor VIIIa and factor X, annexin V reduced the number of binding sites, but also raised the Kd,app. Although factor VIIIa improved the affinity of factor IXa for the lipid surface from Kd approximately 60 nM in its absence to Kd 1 nM in its presence, addition of annexin V to factor IXa titrations on lipid vesicles in the presence of factor VIIIa increased the EC50,FIXa with an IC50 of 1.5 nM, without affecting the Vmax. These data provide evidence that factor IXa, although requiring negatively-charged phospholipid for part of its binding site, is accommodated differently on platelets and on artificial vesicles.

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