Effects of platelets and plasma on fibrinolysis.

The effects of various concentrations of platelets and plasma on in vitro t-PA-induced fibrinolysis were investigated. At t-PA levels between 30 and 70 ng/ml, fibrinolysis proceeded faster in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP). When PRP was serially diluted with PPP and the rate of fibrinolysis compared to PPP, higher platelet counts (up to 300 x 10(9)/l) were associated with increased enhancement of lysis. However, lysis was inhibited when platelets were added to plasma diluted with buffer. Thus, platelets enhance fibrinolysis in undiluted plasma but inhibit lysis in diluted plasma. This is probably because platelets provide a catalytic surface for fibrinolysis but also release inhibitors of fibrinolysis. In undiluted plasma, there would be sufficient fibrinogen and plasminogen to overcome the effect of the platelet inhibitors, but in diluted systems, the inhibitors would predominate, retarding fibrinolysis.     

Enhanced prothrombin-converting activity and factor Xa binding of platelets activated by the alternative complement pathway

Platelet prothrombin-converting activity and factor Xa binding were studied after exposure of human platelet rich plasma (PRP) to various conditions leading to platelet activation. Zymosan resulted in increased platelet-bound C3, enhanced prothrombin-converting activity and increased factor Xa binding. Similar findings were observed with normal platelets resuspended in factor XII-deficient plasma. The combined use of zymosan and thrombin to activate platelets resulted in synergistic prothrombin-converting activity and factor Xa binding. In contrast, no synergism was obtained with the concomitant use of zymosan and collagen, suggesting that collagen and zymosan share the same pathway for platelet activation. Heterologous antibody to factor V completely inhibited the platelet prothrombin-converting activity for all modes of platelet activation, indicating that this activity is mediated by factor V.     

An Examination of Some Factors which Influence the Stability of in Vitro Platelet Responses

Platelet sensitivity to ADP and adrenaline was determined after storage of platelet-rich plasma (PRP) under various conditions to establish those yielding optimal platelet stability. The effects of the exclusion of air from the storage syringes, temperature, PRP dilution and duration of storage were tested. Storage at room temperature (22° C) in the absence of air stabilised PRP pH over 24 h and stabilised platelet sensitivity to ADP up to 4 h. Storage at 4°C and 13 C caused platelet activation and eventually spontaneous aggregation, as evidenced by significant reductions in platelet counts. Samples stored at 37° C were less responsive to ADP and adrenaline than samples maintained at 22 C. Platelet count adjustment to 200 × 10(9)/L reduced platelet sensitivity as reflected by increased agonist EC(50) values and threshold concentrations. Positive correlations between agonist EC(50) values (and between threshold concentrations) for diluted and undiluted samples were obtained, indicating that platelet count adjustment did not affect the ranking order of platelet sensitivity within the subject group. No correlations between platelet count and indices of platelet sensitivity were seen suggesting that differences in platelet aggregation arise from intrinsic differences in platelet sensitivity rather than differences in platelet count. With time of storage the responses to ADP (EC(50) and threshold concentration) and adrenaline (EC(50)) declined to a greater extent for undiluted PRP than for diluted PRP. No changes in the platelet-poor plasma concentrations of the dense granular component, serotonin, occurred in diluted or undiluted samples over 24 h. We conclude that in order to ensure optimal stability of platelets, PRP should be stored at room temperature (22°C) in the absence of air and tested within 4 h of preparation. A decision on platelet count adjustment is also required dependent upon the experimental objectives.     

Enhanced ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib in patients with steroid-responsive nephrotic syndrome

To elucidate the mechanism of enhanced ristocetin-induced platelet aggregation (RIPA) in steroid-responsive nephrotic syndrome (SRNS), plasma levels of von Willebrand factor antigen (vWF:Ag) and ristocetin cofactor (RCof) were examined in 6 patients and the amount of ristocetin-induced vWF binding to platelets was determined. At the initial or relapse stage, the plasma vWF:Ag level was 415 +/- 137% and the RCof level was 364 +/- 117%. The ratio of RCof/vWF:Ag was 0.90 +/- 0.15 and no abnormalities of vWF:Ag multimers were observed, indicating that neither functional nor structural abnormalities were present in patient's plasma. The amount of ristocetin-induced normal vWF binding to nephrotic washed platelets, when ristocetin was used at concentrations of 0.5, 0.75, and 1.0 mg/ml, was 152-163% above the binding to normal platelets. In addition, nephrotic washed platelets resuspended in either normal or nephrotic plasma aggregated at a low concentration of ristocetin (0.75 mg/ml) which did not induce aggregation of normal platelets. In accordance with these observations, the decrease of Alcian blue 8GX binding to platelets, reflecting diminished surface negative charge, was also observed. These results appear to indicate that the plasma vWF level and the altered surface-negative charge in platelets both contribute to heightened vWF binding to GPIb, thus lowering the ristocetin concentration required for RIPA in SRNS.     

An Examination of Some Factors which Influence the Stability of in Vitro Platelet Responses

Platelet sensitivity to ADP and adrenaline was determined after storage of platelet-rich plasma (PRP) under various conditions to establish those yielding optimal platelet stability. The effects of the exclusion of air from the storage syringes, temperature, PRP dilution and duration of storage were tested. Storage at room temperature (22° C) in the absence of air stabilised PRP pH over 24 h and stabilised platelet sensitivity to ADP up to 4 h. Storage at 4°C and 13 C caused platelet activation and eventually spontaneous aggregation, as evidenced by significant reductions in platelet counts. Samples stored at 37° C were less responsive to ADP and adrenaline than samples maintained at 22 C. Platelet count adjustment to 200 × 10⁹/L reduced platelet sensitivity as reflected by increased agonist EC₅₀ values and threshold concentrations. Positive correlations between agonist EC₅₀ values (and between threshold concentrations) for diluted and undiluted samples were obtained, indicating that platelet count adjustment did not affect the ranking order of platelet sensitivity within the subject group. No correlations between platelet count and indices of platelet sensitivity were seen suggesting that differences in platelet aggregation arise from intrinsic differences in platelet sensitivity rather than differences in platelet count. With time of storage the responses to ADP (EC₅₀ and threshold concentration) and adrenaline (EC₅₀) declined to a greater extent for undiluted PRP than for diluted PRP. No changes in the platelet-poor plasma concentrations of the dense granular component, serotonin, occurred in diluted or undiluted samples over 24 h. We conclude that in order to ensure optimal stability of platelets, PRP should be stored at room temperature (22°C) in the absence of air and tested within 4 h of preparation. A decision on platelet count adjustment is also required dependent upon the experimental objectives.     

A new congenital platelet abnormality characterized by spontaneous platelet aggregation, enhanced von Willebrand factor platelet interaction, and the presence of all von Willebrand factor multimers in plasma

A case is reported of a 49-year-old woman with a mild bleeding tendency. Her bleeding time, platelet count and size, plasma ristocetin cofactor activity, von Willebrand factor (vWF) antigen, and vWF multimeric pattern are all within normal limits. Spontaneous platelet aggregation is observed when citrated platelet-rich plasma (PRP) is stirred in an aggregometer cuvette. This aggregation is completely is only slightly diminished by an antiglycoprotein (GP) IIb/IIIa or by an anti GPIb monoclonal antibody. The patient's PRP shows increased sensitivity to ristocetin. The distinct feature of this patient, also present in two family members studied, is that platelet aggregation is initiated by purified vWF in the absence of any other agonist. The vWF- induced platelet aggregation is abolished by anti-GPIb and anti- GPIIb/IIIa monoclonal antibodies and by EDTA (5 mmol/L). Apyrase inhibits the second wave of aggregation. Patient's platelets in PRP are four to six times more reactive to asialo vWF-induced platelet aggregation than normal platelets. The amount of radiolabeled vWF bound to platelets in the presence of either low concentration of ristocetin or asialo vWF was increased 30% compared with normal. The patient's platelet GPIb was analyzed by SDS page and immunoblotting and by binding studies with anti-GPIb monoclonal antibodies showed one band with slightly increased migration pattern and a normal number of GPIb molecules. Unlike the previously reported patients with pseudo or platelet-type von Willebrand disease, this patient has normal vWF parameters.     

Inhibition of ristocetin-induced platelet agglutination by vancomycin.

Ristocetin and vancomycin are structurally similar glycopeptide antibiotics. Both vancomycin and ristocetin in high concentrations (3.0 mg/ml) cause the precipitation of fibrinogen, plasminogen, and IgG from platelet-poor plasma (PPP). In contrast to ristocetin, vanomycin (0.5-1.5 mg/ml) does not agglutinate platelets in normal platelet-rich plasma (PRP) or formalin-treated platelets in the presence of normal PPP. Preincubation of vancomycin (0.5-1.25 mg/ml) with normal PRP, von Willebrand platelets in normal PPP, or formalinized platelets results in inhibition of platelet agglutination induced by ristocetin (0.7-1.25 mg/ml) or ristocetin and normal PPP. This inhibition can be overcome by increasing the final concentration of ristocetin in the platelet suspension. Preincubation of formalin-treated platelets with the major fraction obtained by carboxymethyl-Sephadex C-50 chromatography of commercial vancomycin also results in inhibition of agglutination induced by ristocetin and normal PPP. Incubation with vancomycin (1.25 mg/ml) does not interfere with von Willebrand factor (vWF) or factor VIII coagulant activities in normal PPP or in Sepharose 4B void volume fractions of PPP. These results indicate that vancomycin interacts with normal, von Willebrand, and formalin-treated platelets and inhibits the binding of ristocetin (or ristocetin-vWF complexes).     

Platelet-collagen interaction: inhibition by ristocetin and enhancement by von Willebrand factor-platelet binding

The contribution of von Willebrand factor (vWF)-platelet binding to platelet-collagen interaction was examined in vitro. The binding of vWF to platelets was mediated and regulated by ristocetin. Subthreshold concentrations of ristocetin (less than or equal to 1 mg/mL), insufficient to cause ristocetin-induced platelet aggregation (RIPA), were added to platelet-rich plasma (PRP) prior to the addition of collagen. The collagen-induced platelet aggregation (CIPA) was modified by ristocetin and the degree of alteration was dependent on the ristocetin concentration. Response as a function of ristocetin concentration was designated the Collagen-Platelet Aggregation Response (CoI-PAR). In normal PRP the CoI-PAR was a progressive inhibition followed by decreasing inhibition and then an enhanced response. The enhanced response occurred over a narrow range of ristocetin concentrations (0.8 to 1.0 mg/mL). In the absence of vWF (severe von Willebrand's disease, Type I, vWF less than 1%) the CoI-PAR was a progressive, eventually complete inhibition with no enhanced response (with ristocetin concentrations up to 3.0 mg/mL). With addition of vWF to this PRP an enhanced response was observed at a ristocetin concentration inversely proportional to the vWF level. PRP from a patient with severe Hemophilia A showed a response within the normal range. Subthreshold ristocetin did not cause plasma protein precipitation or platelet release of 3H-serotonin, nor induce micro platelet aggregate formation. Digestion of platelet membrane glycoproteins (GP(s] with chymotrypsin demonstrated that upon removal of GPI, RIPA was absent, CIPA retained and the CoI-PAR was progressive inhibition, with no enhancement. With removal of GPs I, II, and III, RIPA, CIPA, and the CoI-PAR were absent. A dose-response 125I-vWF- platelet binding occurred with increasing ristocetin concentrations which was unchanged by the addition of collagen. These results demonstrated that ristocetin-platelet association inhibited CIPA, and vWF-platelet binding enhanced platelet-collagen adhesion and platelet aggregation. The in vitro-enhanced CIPA represents a vWF-dependent aggregation of sufficient magnitude to overcome the inhibitory effect of ristocetin. These studies demonstrate an influential interaction of ristocetin, vWF, and collagen with the platelet membrane and imply an important hemostatic contribution of vWF-platelet binding in platelet- collagen interaction.     

Acquired von Willebrand disease associated with an inhibitor to factor VIII antigen and gastrointestinal telangiectasia.

A patient with acquired von Willebrand disease and gastrointestinal telangiectasia is described. He presented with the recent onset of spontaneous hemorrhage and demonstrated a prolonged bleeding time, reduced factor VIII coagulant (FVIII:C), and undetectable factor VIII-related antigen (FVIIIR:AG) and ristocetin cofactor (FVIIIR:WF). Following transfusion of cryoprecipitate, there was a smaller than expected immediate increase in FVIII:C, FVIIIR:WF and FVIIIR:AG, with a rapid return to baseline levels and no secondary increase in FVIII:C. An inhibitor could be demonstrated in the patient's plasma which markedly decreased the level of FVIIIR:AG in normal plasma whereas it only weakly decreased the activity of FVIIIR:WF and FVIII:C. The inhibitor was contained in the immunoglobulin G(IgG) fraction of plasma and lacked precipitating properties. This inhibitor demonstrates a specificity not previously seen in spontaneous antifactor VIII antibodies. Our report also demonstrates that ristocetin-induced agglutination of platelets is not always the most sensitive method for detecting an inhibitor in acquired von Willebrand disease.     

血小板聚集初期形状变化与聚集的关系及其影响因素分析

目的探讨血小板聚集初期形状改变与聚集的关系,并分析其影响因素。方法制备小鼠、大鼠富血小板血浆（PRP）和人洗涤血小板,以生理盐水、贫血小板血浆（PPP）或Tyrode液调整血小板计数来建立不同的血小板体系,二磷酸腺苷（ADP）或胶原诱导聚集,光学法测定血小板形状变化指标（最大负值和达最大负值时间）以及最大聚集率。结果人的最大负值、达最大负值时间比小鼠增加（P均〈0.05）,最大负值比大鼠增加（P〈0.01）;大鼠达最大负值时间比小鼠增加（P〈0.01）。生理盐水稀释人PRP比PPP稀释人PRP最大负值减小,达最大负值时间延长（P均〈0.05）。在人PRP中,胶原所致最大负值比ADP减小（P〈0.05）,达最大负值时间增加（P〈0.01）;在人洗涤血小板中,胶原所致最大负值、达最大负值时间均比ADP增大（P均〈0.01）;ADP和胶原在洗涤血小板中比PRP中的最大负值、达最大负值时间（除应用胶原变大外）减小（P均〈0.01）。多元回归分析显示,聚集率与最大负值和达最大负值时间正相关（r分别为0.49、0.48,P均〈0.01）。结论血小板聚集初期变形促进聚集,人的比大鼠、小鼠增强,生理盐水稀释PRP和洗涤血小板减弱,在PRP中胶原比ADP所致变形减弱,在洗涤血小板中增强。     

Effect of heparin on platelet aggregation

The effect of heparin on platelet aggregation was systematically examined on platelets in plasma (PRP), as well as on gel-filtered, washed, and formaldehyde-fixed platelets. Results indicate that, although heparin causes a mild potentiation of platelet aggregation in the PRP systems, a significant inhibitory activity is observed when heparin is added to isolated platelets. This inhibitory activity appears to be specific and not related to the impurities in the heparin preparations, as heparinase, as well as protamine, effectively neutralizes the heparin-mediated inhibitory activity on platelet aggregation. Although heparin-mediated inhibitory activity can be demonstrated in the presence of a number of different agonists (ADP, arachidonic acid, thrombin, Ionophore A23187, epinephrine, and ristocetin), the most pronounced inhibition is seen in the presence of ristocetin. Further studies show that heparin enhances thromboxane generation in isolated platelets. Platelets pretreated with heparin, however, fail to respond to preformed thromboxane. These findings suggest that, in addition to the potentiation of thromboxane production in platelets, heparin may also attribute some change(s) to the platelet(s)/platelet membrane, which interferes with their ability to respond to the agonists of platelet aggregation. This antiaggregatory activity of heparin was found to be inhibited by a factor(s) present in plasma but not in serum.     

Flow cytometry analysis of porcine platelets: optimized methods for best results

Animal models are essential tools for the in vivo evaluation of pharmacological modulation of platelet function and the mechanisms underlying thrombosis. In particular, pigs are being increasingly used in cardiovascular and platelet research. One standard method for the investigation of platelet function under experimental conditions is flow cytometry. However, this approach is limited by a shortage of feasible antibodies and a lack of incubation protocols with regard to porcine platelets. This study aimed to establish a method for the investigation of porcine platelets in flow cytometry. Platelets from pigs and human donors were stained with various commercially available specific antibodies against platelet receptors CD41a, CD42bα, CD62P, activated CD41/CD61, and platelet-bound fibrinogen. Staining procedures were performed in undiluted or diluted whole blood (WB) or platelet-rich plasma (PRP). Samples were treated with PBS buffer as control or with adenosine diphosphate (ADP) to induce platelet activation. Flow cytometry was performed using standard methodology. Furthermore, platelet counts were determined and ADP-induced platelet aggregations of both species were examined to confirm that the agonist ADP reliably activates human as well as porcine platelets. Five of the investigated antibodies bound to human, but not to porcine platelets only. However, two chicken-derived antibodies directed against CD62P (09-143) and fibrinogen (09-038) as well as a monoclonal mouse anti-CD62P (KO2.5) and a polyclonal rabbit anti-fibrinogen antibody (F0111) allowed reliable detection of porcine platelet activation. Moreover, binding intensity of the 09-143 antibody was increased when incubated in porcine PRP compared to WB, whereas antibody binding of both anti-fibrinogen antibodies to porcine platelets was only observed when incubated in a WB-buffer solution. KO2.5 antibody binding was detectable employing PRP as well as the WB-buffer solution. The feasibility of our new incubation protocols was confirmed by successful investigation of platelet activation in a porcine in vivo cardiopulmonary bypass model. In conclusion, we describe a reliable method to detect the activation of porcine platelets and therefore provide a useful tool for platelet flow cytometry in porcine models. Notably, the applied incubation protocol and medium, in which platelets are suspended, have major effects on antibody-binding properties.     

Aurin tricarboxylic acid: a novel inhibitor of the association of von Willebrand factor and platelets

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.     

A plasma inhibitor of ristocetin-induced platelet aggregation in patients with sickle hemoglobinopathies

Because of the high prevalence of thrombotic complications in patients with sickle cell anemia (SCA), we investigated platelet function in patients with sickle hemoglobinopathies. Platelet aggregation induced by epinephrine, ADP, and collagen, except for absent secondary wave in 3 of 10 patients with SCA, was qualitatively normal. However, ristocetin-induced platelet aggregation (RIPA) with a final concentration 1.12 mg/ml was markedly abnormal-absent or virtually absent in 9 of 10 patients with SCA, 3 of 3 patients with hemoglobin S-C disease, and 2 of 3 patients with sickle trait. All 8 controls used in these experiments repeatedly demonstrated normal RIPA. Addition of normal plasma failed to correct abnormal RIPA in sickle hemoglobinopathy patients. All patients demonstrated normal RIPA with a ristocetin dose of 2.24 mg/ml and aggregated with bovine fibrinogen. Recombinant mixing experiments demonstrated that washed SCA platelets support RIPA (1.12 mg/ml) when resuspended in normal plasma or high dilutions of SCA plasma, but not in undiluted SCA plasma. Washed normal platelets do not support RIPA (1.12 mg/ml) when resuspended in SCA plasma. These findings suggest the presence of a plasma inhibitor of RIPA in patients with sickle hemoglobinopathies.     

Evidence that Endotoxins Enhance the Factor X Activator Activity of Washed Human Platelets

The in vitro effect of several bacterial endotoxins on human platelets was determined. Nine different endotoxins failed to induce aggregation in platelet-rich plasma (PRP) or of platelets washed by two different methods; four of them which we studied further failed to induce [14C]serotonin release in PRP. In contrast, using recently described test systems for platelet coagulant activity, all the endotoxins shortened the latent period occurring before aggregation of a mixture of washed platelets, normal serum, and CaCl2, and the clotting time of this mixture upon addition of fibrinogen. Washed platelets obtained from PRP preincubated with endotoxin had a higher platelet coagulant activity than platelets obtained from PRP preincubated with buffer. Washed platelets contribute to thrombin generation by providing factor V, a factor X activator and possibly phospholipid. Since the endotoxins did not influence the factor V activity of platelets or the platelet factor 3 activity, either in PRP or using platelets washed by albumin density gradient centrifugation, it is suggested that they enhance the factor-X activator activity of human platelets.     

The effect of oxidatively modified low-density lipoproteins on platelet aggregability and membrane fluidity

The influence of oxidised low-density lipoproteins (oxLDL) on blood cell functions plays a role in the progression of atherosclerosis. In the present work the effects of mildly oxidised LDL (moxLDL) on platelet aggregability and plasma membrane fluidity were studied and analysed from the viewpoint of the extent of LDL oxidation. Native or oxidised LDL were incubated with platelet rich plasma (PRP) at the volume ratio 1:1. As a control, plasma was incubated with buffer. The effects on ADP-induced platelet aggregation and certain membrane characteristics are described. (1) Mildly oxidised LDL diminished the time-dependent decrease in platelet aggregability that was observed when PRP was incubated with buffer or native LDL. The higher the oxidation extent of moxLDL, the lesser (if any) decrease in platelet activity occurred. Therefore moxLDL activated platelets in PRP. Cu2+-oxidised LDL, characterised by a high extent of lipid oxidation, inhibited ADP-induced platelet aggregation. (2) Comparison of the ESR spectra of spin-labelled fatty acid (5-doxylstearate) incorporated into the plasma membrane of washed platelets indicated that the presence of moxLDL in the incubation medium resulted in a reduced fluidity of the outer membrane layer. The cholesterol:phospholipid ratio in platelets appeared to be the same after PRP incubation with native LDL, moxLDL or buffer. It may be proposed that the binding of oxLDL to the platelet surface leads to a modification of the membrane fluidity, thus mediating the activating action of LDL on platelets. Both effects were proportional to the extent of lipid oxidation in LDL. The results of this paper indicate a crucial role for mildly oxidised LDL in platelet activation.     

Botrocetin- and polybrene-induced platelet aggregation in platelet-type von Willebrand disease

It has been reported that botrocetin, a Bothrops venom factor, induces platelet aggregation dependent on von Willebrand factor (vWF), and that platelet aggregation induced by Polybrene, a synthetic polycation, is enhanced by vWF. This report describes the platelet aggregability on stimulation with botrocetin and Polybrene in four patients with platelet-type von Willebrand disease (vWD) who showed increased platelet aggregation with low concentrations of ristocetin as the result of a platelet abnormality. Enhanced platelet aggregability with botrocetin was observed in platelet-rich plasma (PRP) from the patients. Platelet aggregation induced by botrocetin in a mixture of normal washed platelets and patient plasma was either decreased or normal, being dependent on the amount of plasma vWF. In contrast with ristocetin and botrocetin, Polybrene did not cause increased aggregation of patient PRP. Polybrene aggregated normal washed platelets less extensively in the presence of patient plasma than normal plasma. These studies demonstrated that botrocetin induced heightened interaction between platelets and vWF, but Polybrene did not, in platelet-type vWD, and that the enhanced responsiveness of patient platelets to botrocetin is related to an intrinsic platelet abnormality.     

The pH dependence of quantitative ristocetin-induced platelet aggregation: theoretical and practical implications-a new device for maintenance of platelet-rich plasma pH.

Quantitative ristocetin-induced platelet aggregation of normal platelet-rich plasma (PRP) decreased with time after PRP preparation. An increase in p H of the PRP with time proved to be responsible for this finding. Diffusion of CO2from the plasma is the prime determinant of the change in pH. Since a complex combination of factors influences CO2 diffusion (surface area-to-volume relationship, capping, mixing, etc.) The change in pH is variable with time. Thus, quantitative ristocetin aggregation should be pH controlled. A simple device for maintaining PRP pH constant by control of the ambient pCO2 was designed and found effective in keeping both pH and quantitative ristocetin aggregation constant over a prolonged period of time. It can be adapted for use in platelet aggregation studies employing other reagents. The pH dependence of ristocetin-induced platelet aggregation is consistent with other data supporting an elctrostatic interaction between the platelet, von Willebrand factor, and ristocetin. We favor a model wherein ristocetin neutralizes some of the platelet's negative change and permits the von Willebrand factor to bridge sites on separate platelets to induce agglutination.     

The agglutination of human platelets by botrocetin: evidence that botrocetin and ristocetin act at different sites on the factor VIII molecule and platelet membrane

S ummary. Botrocetin caused a factor VIII (FVIII) dependent platelet agglutination which was associated with a reduction in the plasma levels of all FVIII parameters as a result of specific binding of FVIII to the platelets. The site of binding of FVIII to the platelet in response to ristocetin or botrocetin involves the glycoprotein 1 complex.
This is suggested by the inability of chymotrypsin treated platelets or platelets from patients with the Bernard-Soulier syndrome to agglutinate in response to ristocetin.
These platelets responded to botrocetin, but this was greatly reduced compared to normal. Crossed immunoelectrophoretic analysis indicated that in the presence of botrocetin most multimetric forms of FVIII bound to the platelet, whereas ristocetin caused binding of high and intermediate molecular weight forms. The antibiotic vancomycin inhibited platelet agglutination by ristocetin but had no effect on that caused by botrocetin. Assays of FVIII von Willebrand factor (VIII:vWf) using botrocetin compared well with those obtained using ristocetin in plasmas from normal individuals and from patients with classical von Willebrand disease (vWd).
However, a patient with variant vWd demonstrated 100% botrocetin cofactor activity and 0% ristocetin cofactor activity. This suggested that the site of interaction on the FVIII molecule for botrocetin and ristocetin are different. Therefore the diagnosis of some von Willebrand variants cannot be excluded on the basis of a normal botrocetin cofactor assay.     

The effect of oxidatively modified low-density lipoproteins on platelet aggregability and membrane fluidity

The influence of oxidised low - density lipoproteins (oxLDL) on blood cell functions plays a role in the progression of atherosclerosis. In the present work the effects of mildly oxidised LDL (moxLDL) on platelet aggregability and plasma membrane fluidity were studied and analysed from the viewpoint of the extent of LDL oxidation. Native or oxidised LDL were incubated with platelet rich plasma (PRP) at the volume ratio 1:1. As a control, plasma was incubated with buffer. The effects on ADP - induced platelet aggregation and certain membrane characteristics are described. (1) Mildly oxidised LDL diminished the time - dependent decrease in platelet aggregability that was observed when PRP was incubated with buffer or native LDL. The higher the oxidation extent of moxLDL, the lesser (if any) decrease in platelet activity occurred. Therefore moxLDL activated platelets in PRP. Cu2 + -oxidised LDL, characterised by a high extent of lipid oxidation, inhibited ADPinduced platelet aggregation. (2) Comparison of the ESR spectra of spin - labelled fatty acid (5 - doxylstearate) incorporated into the plasma membrane of washed platelets indicated that the presence of moxLDL in the incubation medium resulted in a reduced fluidity of the outer membrane layer. The cholesterol:phospholipid ratio in platelets appeared to be the same after PRP incubation with native LDL, moxLDL or buffer. It may be proposed that the binding of oxLDL to the platelet surface leads to a modification of the membrane fluidity, thus mediating the activating action of LDL on platelets. Both effects were proportional to the extent of lipid oxidation in LDL. The results of this paper indicate a crucial role for mildly oxidised LDL in platelet activation.