Factor VIIIa regulates substrate delivery to the intrinsic factor X‐activating complex

Abstract

Activation of coagulation factor X (fX) by activated factors IX (fIXa) and VIII (fVIIIa) requires the assembly of the enzyme-cofactor-substrate fIXafVIIIa-fX complex on negatively charged phospholipid membranes. Using flow cytometry, we explored formation of the intermediate membranebound binary complexes of fIXa, fVIIIa, and fX. Studies of the coordinate binding of coagulation factors to 0.8-lm phospholipid vesicles (25 ⁄ 75 phosphatidylserine ⁄ phosphatidylcholine) showed that fVIII (fVIIIa), fIXa, and fX bind to 32 700 ± 5000 (33 200 ± 14 100), 20 000 ± 4500, and 30 500 ± 1300 binding sites per vesicle with apparent K d values of 76 ± 23 (71 ± 5), 1510 ± 430, and 223 ± 79 nm, respectively. FVIII at 10 nm induced the appearance of additional high-affinity sites for fIXa (1810 ± 370, 20 ± 5 nm) and fX (12 630 ± 690, 14 ± 4 nm), whereas fX at 100 nm induced high-affinity sites for fIXa (541 ± 67, 23 ± 5 nm). The effects of fVIII and fVIIIa on the binding of fIXa or fX were similar. The apparent Michaelis constant of the fX activation by fIXa was a linear function of the fVIIIa concentration with a slope of 1.00 ± 0.12 and an intrinsic K m value of 8.0 ± 1.5 nm, in agreement with the hypothesis that the reaction rate is limited by the fVIIIa-fX complex formation. In addition, direct correlation was observed between the fX activation rate and formation of the fVIIIa-fX complex. Titration of fX, fVIIIa, phospholipid concentration and phosphatidylserine content suggested that at high fVIIIa concentration the reaction rate is regulated by the concentration of free fX rather than of membrane-bound fX. The obtained results reveal formation of high-affinity fVIIIa-fX complexes on phospholipid membranes and suggest their role in regulating fX activation by anchoring and delivering fX to the enzymatic complex.Abbreviations BSA, bovine serum albumin; DiIC16(3), 1,1¢-dihexadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate; fVIII(a),

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting

“…When the same samples were observed with confocal microscopy ( Figure 1B), we did not see any significant binding of Annexin V to the nonprocoagulant spread platelets. 5,21 In contrast, Annexin V binding to the balloonshaped platelets was very pronounced. It could be seen that the "cap" appeared as a small bright region in the Annexin V channel, indicating that it is enriched with PS (supplemental Video 1).…”

“…FVIIIa was assumed to be bound to the surface due to its small dissociation constant. 22 The vesicle radius was considered to be 400 nm 21 and the platelet radius assessed in the current study was 2000 nm as discussed later. The diffusion constants for all bound factors were assumed to be 0.001 mm 2 per second.…”

“…Computer model of intrinsic tenase assembly and activation on the phospholipid membrane The model was designed based on the binding and kinetic experimental data as described in Panteleev et al 21 Its development, design, and validation are outlined in supplemental Figure 1. The resultant model consisted of 7 partial differential equations, and described the interaction of FX and FIXa with phospholipid membranes, assembly of intrinsic tenase on their surface, and activation of FX by the complex.…”

“…21 Phospholipids were transferred to a round bottom flask and dried for 30 minutes under nitrogen. They were hydrated for 30 minutes in 20 mM HEPES, 140 mM NaCl, pH 7.5 for T .50°C.…”

Thrombin generation, fibrin clot formation and hemostasis

“…68 These findings suggest that thrombin generated on the surface of platelets during the propagation phase dictates that rate of fibrin clot formation away from the initiating cells. [67][68][69][70] Fibrin formation during cell-mediated thrombin generation likely includes a complex spatial component as well. It is likely that this physical progression of procoagulant activity from the tissue factor-bearing cell (low levels of thrombin generation) to the activated platelet surface (rapid burst of thrombin generation) induces the formation of a thrombin gradient in space.…”