Plateletpheresis by discontinuous centrifugation: effect of collecting methods on the in vitro function of platelets.

The in vitro function of platelets collected by two different methods during centrifugal plateletpheresis was compared. The RBC method involves collecting platelets with red cells followed by a supplementary spin to remove them, whereas the no-RBC method requires collecting platelets only from the buffy coat without red cells. Platelet response to adenosine diphosphate (ADP), epinephrine and collagen was slightly reduced in platelet-rich plasma (PRP) prepared by no-RBC technique and was markedly decreased in samples obtained by the RBC technique when compared to prepheresis controls. The decrease in platelet response to ADP, epinephrine and collagen was apparent in three testing systems: aggregation, release of serotonin and reptilase clot retraction. Both plasma and platelets appeared to be affected by the pheresis procedure. Platelet preparations obtained by both RBC and no-RBC techniques showed an increase of platelet factor 3 activity and an enhancement of aggregation, release of serotonin and clot retraction induced by thrombin as compared to prepheresis controls. Postpheresis platelet-poor plasma contains platelet membrane fragments which exhibit a high platelet factor 3 activity. The results showed that the RBC method, although providing a higher platelet yield, caused more qualitative alterations in platelets than in those obtained by no-RBC method, and that both methods of collecting platelets activated the procoagulant activity of platelets.

Aggregation of platelets and inert particles induced by thrombin.

Thrombin-induced platelet aggregation and release were investigated in washed platelet suspensions and in suspensions of inert particles in order to evaluate the role of fibrinogen-fibrin transformation in aggregometer tracings. Thrombin (0.25-2.0 U/ml) produced two waves of light transmission increase (LTI) in both platelet and inert particle suspensions containing fibrinogen, and concomittantly aggregates were observed under phase microscopy. Without fibrinogen, thrombin induced rapid release of platelet ADP but failed to cause second wave of LTI. The kinetics of LTI in platelet and inert particle systems were related to both thrombin and fibrinogen concentrations. A rapid second wave of LTI could be produced by direct interaction of thrombin-treated platelets or inert particles with polymerizing fibrin, and was inhibited by sodium sulfite and low pH of 5.1 which prevent fibrin monomer polymerization. No fibrin strands were noted in platelet aggregates fixed at the completion of the second wave of LTI. Apyrase and PGE1 inhibited the rate of first but not that of second wave LTI. The results suggest that the release of platelet ADP induced by thrombin primarily affects the first phase aggregation, and the second phase may result from interaction of thrombin-exposed platelets and polymerizing fibrin. Thus, the blood coagulation mechanism may be directly involved in platelet aggregation.

Colchicine uptake and binding by human platelets.

The uptake and binding of antimitotic alkaloid colchicine has been demonstrated in washed preparations of human pletelets. A silicone oil technique was adapted so that both uptake and binding of 14C-colchicine were examined in the same platelet preparations. The time dependence and amount of colchicine taken up and bound by different pletelet preparations during a 90 to 120 min incubation period were highly reproducible. Both colchicine uptake and binding by intact platelets, and colchicine binding by preparations of lysed platelets were specific and temperature dependent. Colchicine uptake was slowly reversible. Magnesium and GTP enhanced colchicine binding by lysed platelet preparations but calcium decreased binding. exposure of platelets to either cold (4 degrees C) or to thrombin, which disrupt platelet microtubules, produced significant increases in colchicine uptake and binding. The thrombin effect was maximal at 37 degrees C and resulted in a greater increase in uptake and binding than that produced by either cold treatment alone or, by cold treatment followed by incubation with thrombin at 37 degrees C. The amount of increase in uptake and binding produced by thrombin was independent of both thrombin (1--5 Units/10(9) platelets) and colchicine concentrations (1--50 X 10(-6) M). It is postulated that thrombin may initiate the formation, or make available, colchicine binding sites (microtubule subunits) within platelets.

Concentration effects of platelets, fibrinogen and thrombin on platelet aggregation and fibrin clotting.

The effects of varying concentrations of platelets, fibrinogen and thrombin on platelet aggregation and on fibrin clotting were investigated. The results indicated that a threshold thrombin to platelet concentration ratio may be required to cause platelet activation. Above the threshold ratio, platelets exhibited properties which enhanced thrombin action in causing aggregation and fibrin clotting. At T/P ratios below the threshold level, the presence of platelets reduced thrombin activity, in other words, platelets exerted an antithrombin action. Fibrinogen at low concentrations (0.02-1.5 mg/ml) enhanced platelet aggregation induced by thrombin; whereas, at high concentrations of fibrinogen (2.0-4.0 mg/ml), aggregation was markedly inhibited. Continuous mixing of samples of paltelets and fibrinogen at physiological concentrations with thrombin at low concentrations (less than 2.0 U/ml) resulted in platelet aggregation. On the other hand, fibrin clots formed in samples without mixing or with high thrombin concentrations (greater than or equal to 5.0 U/ml). These results suggested that the quantitative relationships between platelets, fibrinogen and thrombin, and the presence or absence of cell contact may be important factors in determining the overall hemostasis.