Some Effects of Calcium on the Activation of Human Factor VIII/Von Willebrand Factor Protein by Thrombin

Abstract

A B S T R A C T When Factor VIII/von Willebrand factor (FVIII/vWF) protein is rechromatographed on 4% agarose in 0.25 M CaCl2, the protein and vWF activity appear in the void volume, but most ofthe FVIII procoagulant activity elutes later. Recent evidence suggests that the delayed FVIII procoagulant activity is a proteolytically modified form of FVIII/vWF protein that filters anomalously from agarose in 0.25 M CaCl2. To test whether or not thrombin is the protease involved, the effect of 0.25 M CaCl2 on FVIII/vWF and its reaction with thrombin was examined. About 30% of the FVIII procoagulant activity was lost immediately when solutions of FVIII/vWF protein were made 0.25 M in CaCl2. When FVIII in 0.15 M NaCl was activated with 0.04 U thrombin/ml and then made 0.25 M in CaCl2, the procoagulant activity of a broad range of FVIII/vWF protein concentrations remained activated for at least 6 h. But, in 0.25 M CaCl2, the increase in FVIII procoagulant activity in response to thrombin was much more gradual and once activated, the procoagulant activity was stabilized by 0.25 M CaCl2. When thrombin-activated FVIII/vWF protein was filtered on 4% agarose in 0.15 M NaCl, there was considerable inactivation of FVIII procoagulant activity; however, the procoagulant activity that did remain eluted in the void volume. In contrast, when thrombin-activated FVIIIVvWF protein was filtered in 0.25 M CaCl2, the FVIII procoagulant activity eluted well after the void volume and remained activated for 6 h.

Influence of high molecular weight factor VIII on the measurement of low molecular weight factor VIII procoagulant in different assay systems

“…The CaCll concentration in the elution buffer is crucial in regard to the elution pattern and the stability ofLMW VIIIC. With a low CaClz concentration, a labile LMW VIIIC is obtained; high CaClz concentrations in the elution buffer result in one peak of LMW VIIIC with very stable activity (Tuddenham et al, 1979). Our results are in close correlation with these observations.…”

Calcium Prevents Enhanced Degradation of Factor VIII in the Condition of Motion

“…We have also demonstrated a protective effect of factor VIII on factor IX level, and vice versa [8]. Calcium ions had earlier been reported to stabilize both native and thrombin-activated factor VIII-von Willebrand factor (vWF) complexes using in vitro assays [9,10]. To that end, the present study aimed to explore if the stabilization effect of calcium on factor VIII persists in the condition of motion, and if oral supplementation of calcium to mice could prevent enhanced factor VIII degradation during movement.…”

Interaction of factor VIII-von Willebrand Factor with phospholipid vesicles

“…This is supported by various experiments demonstrating separation between Factor VIII activity and von Willebrand Factor-related protein (Weiss et al, 1972;Weiss & Hoyer, 1973;Cooper et al, 1973;Baugh et al, 1974;Holmberg & Ljung, 1978;Horowitz et al, 1979). It has been questioned (Switzer & McKee, 1976, 1977, however, if those separations obtained are not the result of limited proteinase action cleaving off the Factor VIII activity part from a Factor VIII-von Willebrand Factor molecule. This opinion has been supported by observations that the separation of Factor VIII activity from von Willebrand Factor protein is influenced by the presence of proteinase inhibitors, less separation being obtained in the presence of proteinase inhibitors (Beck et al, 1976).…”

“…The Km for the activation reaction is very low (Broden et al, 1980), suggesting that thrombin has a very high affinity for Factor VIII. The activation by thrombin has also been shown to result in separation of Factor VIII activity and Factor VIIIRAg (Switzer & McKee, 1977).…”