Robert A. Grant

Binding of factor VIII to platelets in the presence of ristocetin.

Ristocetin agglutinates platelets in platelet‐rich plasma (PRP) containing factor VIII‐related von Willebrand factor (WF). When the clumps were separated from the supernatant and resuspended in buffer, the platelets dispersed if the buffer was free of ristocetin and the PRP had been treated with aspirin or EDTA to prevent the platelet release reaction. Binding to platelets was determined by measuring WF (ristocetin cofactor assay), VIIIR‐antigen (AG, by radioimmunoassay), and coagulant activity (C) in the initial plasma, the supernatant remaining after removing the clumps formed by ristocetin, and the eluate produced after resuspending the clumped platelets in buffer. No activity was bound to platelets in the absence of ristocetin. After agglutinating platelets in PRP with ristocetin, WF, AG and C activities in the supernatants were about 65% of the initial plasma values, and in the eluates about 9%. Formalin‐fixed washed platelets resuspended in 1 in 3 plasma, and platelets in PRP diluted 1 in 3 also agglutinated when shaken with ristocetin. There was no difference between results with fixed and unfixed platelets. In one series of experiments, the supernatants had about 60% of the initial WF and AG activities; since these activities were the same, they may measure the same substance. In a later series, WF and C were compared; in shaken samples the supernatants had 39% of the initial WF activity and 56% of the initial C activity, and in unshaken samples the supernatants had 3% WF and 29% C. The significantly lower values for C binding suggest that this activity is located on a different molecule from WF‐AG and that the molecules are more readily separated in diluted than in undiluted plasma. Activity in the eluates was inversely proportional to that in the supernatants although the initial activity was never completely recovered. WF and C binding increased with platelet number and ristocetin concentration. Platelets exposed to ADP before fixation showed less agglutination than control fixed platelets and bound less WF. Fixed platelets incubated for 15 min with 30 μg trypsin/ml and washed, or platelets from a patient with the Bernard‐Soulier syndrome failed to agglutinate and bound virtually no WF, AG or C. We conclude that WF, AG and C reversibly bind to fixed or unfixed platelets in the presence of ristocetin, that binding is similar in undiluted plasma but greater for WF and AG than C in plasma diluted 1 in 3, and that ADP and trypsin alter binding, probably by affecting a platelet receptor.

Decreased Association of 45Calcium with Platelets Unable to Aggregate due to Thrombasthenia or Prolonged Calcium Deprivation

45Calcium uptake was studied with aspirin‐treated platelets that were gel‐filtered through a column of Sepharose 2B equilibrated with divalent cation‐free modified Tyrodes solution to remove readily exchangeable surface‐associated calcium. These platelets aggregated almost immediately when exposed to ADP, fibrinogen and at least 30 μm CaCl2. At this calcium ion concentration, 108 platelets took up 36.6 ± SEM 2.7 pmol of 45calcium within 1–2 min. The presence of ADP and fibrinogen did not affect the amount of calcium bound. Over 90% of this platelet‐associated calcium was removed by EDTA in 5 min suggesting that it was surface‐bound. Calcium uptake increased rapidly for 10 min, then more slowly for up to 2 h. At 60 min, maximal uptake was approached at CaCl2 concentrations between 250 and 300 μm when an average of 276 ± SEM 18 pmol of calcium was associated with 108 platelets. Only 50–60% of this calcium could be removed by EDTA in 5 min, and about 70% in 20 min, suggesting that some of it had been internalized. Platelets from two patients with thrombasthenia that were unable to aggregate took up 50% less calcium than platelets from normal volunteers. Similarly, platelets that had been incubated with EDTA at 37°C, pH 7.8 for 8 min lost the ability to aggregate despite recalcification and took up 40–60% less calcium than CaEDTA‐treated controls. Platelets from a patient with the Bernard‐Soulier syndrome aggregated and bound calcium normally. Thus the platelets’ ability to take up calcium after removal of surface‐associated calcium correlates with their ability to aggregate. Since thrombasthenic platelets and platelets rendered incapable of aggregating after prolonged calcium deprivation with EDTA do not bind fibrinogen, we postulate that some of the surface‐associated calcium normally binds to the fibrinogen receptors.

Effects of Chymotrypsin on the Release Reaction and Aggregation of Blood Platelets

Abstract Platelets incubated with chymotrypsin and washed are known to aggregate with fibrinogen. Chymotrypsin is reported to cleave platelet membrane glycoproteins without causing the release reaction seen with some proteolytic enzymes such as trypsin. Recent batches of chymotrypsin from two manufacturers caused platelets to release much of the contents of their dense granules, largely because they are contaminated with trypsin. However, even after inhibition of trypsin with N-α-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), chymotrypsin induced some release from platelets at low concentrations of ionized calcium. As expected, platelets incubated with either trypsin-contaminated or TLCK-treated chymotrypsin aggregated with fibrinogen after they were washed. Released or added ADP enhanced the release reaction and development of aggregability with fibrinogen. Thus, [14C]serotonin secretion and fibrinogen-induced aggregation were less when released ADP was enzymatically destroyed during incubation of platelets with 500 μg/ml of trypsin-contaminated chymotrypsin, and they were increased if 25 μM ADP was added before incubating the platelets with 125 μg/ml of this chymotrypsin.