Metabolism and Function of Human Platelets Washed by Albumin Density Gradient Separation

A method for washing platelets by albumin density gradient separation, originally designed for the study of platelet coagulant activities, has been modified for platelet aggregation and metabolic studies. Platelets are sedimented into a continuous density gradient of isosmolar albumin containing apyrase to protect them from clumping and physical injury and are resuspended in calcium-free Tyrode's solution. The mean recovery of platelets after two separations relative to plateletrich plasma (PRP) was 90.3%. When small amounts of plasma were added to washed platelet suspensions, aggregation and release of [¹⁴C]5-hydroxytryptamine (5HT) in response to adenosine diphosphate (ADP) or 5HT were similar to results obtained with PRP. When fibrinogen was substituted for plasma, ADP-induced aggregation occurred but was feeble. Without added plasma or fibrinogen, platelets were refractory to ADP and insensitive to the cyclic endoperoxide analogue U44619. When both ADP and U44619 were added simultaneously, in low concentrations, to washed platelets without added plasma or fibrinogen, aggregation occurred immediately. Washed platelets were not aggregated by adrenaline, which potentiated ADP-induced aggregation. Several biochemical measurements which are sensitive indicators of cellular damage were normal in washed platelets, including [¹⁴C]adenine uptake, adenylate energy charge, hypoxanthine formation and the response of adenylate cyclase to stimulation by PGE₁ or PGD₂. Platelet coagulant activities were not made available and heparin-neutralizing activity (HNA) was not spontaneously released by the washing procedure, but the washed platelets responded normally to appropriate agents by developing coagulant activities and releasing HNA. The ultrastructure of washed platelets was similar to those in control PRP. Inclusion of apyrase in the first albumin gradient had a beneficial effect on platelet morphology, aggregation and metabolism, but washing at 37deg;C compared with 25deg;C did not. Albumin density gradient separation is a useful method for isolating platelets for aggregation and metabolic studies.     

Platelet Coagulant Activities in Thrombasthenia

S ummary . Methods for assaying four platelet coagulant activities are presented. The results of performing these assays in six patients with thrombasthenia revealed decreased contact-forming activity (CPFA) and an absence of platelet response to adenosine diphosphate in the CPFA assay. All patients had normal or raised intrinsic factor-Xa forming activity (XaFA) and decreased platelet factor-3 activity (PF₃A), the level of which related poorly to bleeding history. The four patients most severely affected clinically lacked collagen-induced coagulant activity (CICA), whereas CICA was present in the other two. The important determinants of bleeding severity in thrombasthenia may be the platelet coagulant activities concerned with the initiation of intrinsic clotting (i.e. CICA and CPFA) and not those concerned with the subsequent interaction of clotting factors other than the contact factors (i.e. XaFA and PF₃A). It is concluded that each of the four coagulant activities studied is separate and distinct from the others, and each has its own biochemical determinants.     

Comparison of the Coagulant Activities of Platelets and Phospholipids

The coagulant activities of various phospholipid preparations were compared with those of platelets. Folch phospholipid with maximal platelet factor 3 (PF3) activity produced long recalcified clotting times of relatively undiluted plasma in plastic tubes whereas untreated or ADP-treated platelets with minimal PF3 activity produced short clotting times in the same test system which is sensitive to activators of the contact system of intrinsic coagulation. Bell and Alton phospholipids with maximal PF3 activity produced recalcified clotting times similar to those in the presence of platelets. Bell and Alton phospholipids had tissue factor activity, but Folch phospholipid and platelets did not. Bell and Alton phospholipids and gum acacia (used as a vehicle in one of the preparations) activated factor XII as did platelets, but Folch phospholipid did not. The multiple coagulant activities of Bell and Alton phospholipids (i.e. PF3, tissue factor and contact activating) may account for the absence of coagulant superiority of platelets in the undiluted system in plastic tubes. The coagulant activities of platelets are also complex but different from Bell and Alton phospholipids whereas Folch phospholipid would appear to possess only PF3 activity.     

Platelet Coagulant Activities and Hemostasis: A Hypothesis

Platelets have recently been shown to participate in reactions with blood coagulation factors at every stage of intrinsicclotting, from contact activation to fibrinformation. Platelets can trigger intrinsiccoagulation by two alternative pathways,the first of which involves factors XII andXI and adenosine diphosphate, and thesecond of which bypasses factor XII, provided factor XI and collagen are present.Additional evidence indicates that subsequent coagulation reactions occur on theplatelet surface, where active clottingfactors are protected from inactivation bynaturally occurring inhibitors. Based onthese observations, an hypothesis is presented in which the events of primaryhemostasis (platelet adhesion, aggregation, and release) and blood coagulationare linked. As platelets aggregate to forma hemostatic plug, they provide a protective and catalytic surface for activation of the clotting mechanism and fibrinformation. Localized hemostasis is promoted and circulating blood kept fluid bymeans of a number of control mechanisms,some of which are mediated by autocatalytic effects of thrombin.

Accepted on October 25, 1973     

The Coagulant Activity of Platelets

When platelet-rich plasma is incubated for 16–20 hours at 37° C. and the platelets are then separated and washed, tissue-factor activity develops in these platelets. The active platelets accellerate the clotting of plasma samples deficient in Factor XII, XI, IX or VIII, and of normal plasma; the coagulant activity for samples deficient in Factors V, VII and X is much less marked. The activity developed will cause activation of Factor X in a serum eluate, whereas no such activity is evident in platelets separated from plasma immediately after collection from the donor.
The development of tissue-factor activity of platelets depends on the presence of Factor XII but not on the presence of any of the other factors tested. The relationship of tissue-factor activity to platelet factor 3 is discussed.     

Platelets in Myeloproliferative Disorders. III: Glycoprotein Profile in Relation to Platelet Function and Platelet Density

Membrane glycoproteins (GP) are implicated in platelet functions. In myeloproliferative diseases (MD), some of these functions are known to be perturbed. 16 patients with various MD were investigated for platelet functions (retention to glass beads, epinephrine- and ristocetin-induced aggregation), platelet density distribution and PAS-staining glycoprotein profile on SDS-polyacrylamide-gel-electrophoresis. An abnormal GP pattern (moderate reduction of GP (Ib + Is) and GP IIb with corresponding increase in GP IIIb) was demonstrated but no relation to platelet dysfunction or density distribution was observed. No differences between the various types of MD were noticed although the group of poly-cythaemia vera was the less perturbed for platelet function, platelet density and also GP profile.     

Density Gradient Separation of Lymphoid Cells Adhering to Protein-A-Containing Staphylococci

A cell separation technique was designed based on the interaction between cell-surface-bound IgG and protein A of Staphylococcus aureus. The density of lymphoid cells coated with IgG antibodies against one of the surface markers was increased by adherence of staphylococci. Cells with adhering bacteria were separated from cells without bacteria by density gradient centrifugation in 11.5% sodium metrizoate. Bacteria were removed from the lymphoid cells by lysostaphin digestion.The purity of separated cells was approximately 95% even when the proportion of a specific cell population was below 10% in the initial mixture. The viability and the ability of cells to multiply in vitro were not significantly impaired by the fractionating procedure.The technique can generally be applied for cell separation, provided antibodies of the IgG class against specific surface markers are available.     

The Effect of Dilution of Plasma on Coagulation: The Significance of the Dilution-Activation Phenomenon for the Study of Platelet Coagulant Activities

Summary: The coagulation of non-contacted plasma is enhanced by dilution with citrate-saline solution, an effect termed the 'dilution-activation phenomenon (DAP)'. When platelets rather than phospholipid were added to plasma, the dilution effect was greatly attenuated. A large clotting time difference between platelet and phospholipid additions was therefore evident when undiluted plasma was tested in plastic tubes. This difference disappeared when the plasma was diluted and tested in glass tubes. Thereby the'DAP'masked a platelet coagulant activity which appeared to be distinct from platelet factor 3 activity.
In order to make meaningful use of a test system for platelets which eliminated the 'DAP', it was desirable to ascertain what factors accounted for the clot-promoting effect of dilution. The experimental evidence suggests that a physicochemical alteration results in factor-XII activation when plasma is diluted. Physicochemical factors shown to be important in enhancing the'DAP'and presumably contact activation are alkaline pH, low ionic strength and negative electrochemical charge, whereas positive charge, high ionic strength and acid pH inhibit clotting in the system employed.     

Platelets and Platelet Phosphatides in Uremia

1. The platelets of 20 uremic subjects were studied, utilizing the followingprocedures; phase platelet count, serum prothrombin time, thromboplastingeneration test with evaluation of plasma, serum, and platelet reagents.

2. Platelet phospholipids from these subjects were used as reagents in thethromboplastin generation test and examined by means of silicic acid paperand column chromatography.

3. Thrombocytopenia was the most common abnormality encountered andwas associated with either normal or abnormal prothrombin consumption. Somepatients demonstrated defective prothrombin utilization despite normal platelet counts, but their platelets had normal thromboplastic activity, as did thoseof all patients studied.

4. The paper chromatographic pattern of the phosphatides in all subjects wasthe same as that reported for normal platelets. Similar results were obtainedon column chromatographic analysis of a pooled extract from nine of theuremic patients.

5. On the basis of these studies it was not possible to demonstrate a qualitative platelet defect in uremia.

Submitted on February 8, 1960 Accepted on May 4, 1960     

Platelets and Platelet Inhibitors in Infective Endocarditis

The pathogenesis of infective endocarditis depends on complex interactions between the causative pathogen, plasma proteins, platelets, and vascular endothelial cells. In addition to being the main target of bacteria in the initial stage of bacterial adherence to the endocardium, platelets now appear to play an important role in antimicrobial host defense against endocarditis through the secretion of socalled platelet microbicidal proteins. In animal models of endocarditis, low-dose aspirin was shown to significantly reduce the vegetation weight, the bacterial density of vegetation, the hematogenous bacterial dissemination, and the frequency of embolic events. However, these facts cannot be extrapolated to clinical care in humans, since to date, there is no definitive proof of the adjunctive benefit of aspirin in human infective endocarditis.     

How Platelets Work: Platelet Function and Dysfunction

This article briefly reviews (a) how platelets normally function and (b) the clinical approach to disorders of platelet numbers and function.     

Aggregation of Human Platelets by Bovine Platelet Fibrinogen

Aggregation of human platelets by addition of purified bovine platelet fibrinogen is described. Bovine plasma fibrinogen showed the same but much weaker effect. Human fibrinogen produced no aggregation. No absolute requirement for divalent cations or plasma proteins could be demonstrated. The aggregation of washed platelets appeared monophasic whereas in platelet-rich plasma it was usually biphasic. The use of inhibitors of ADP-induced platelet aggregation, inhibition of intracellular ATP-production, enzyme-catalyzed removal of ADP, and direct determinations of ADP in the medium showed the second phase to be mediated by ADP released from the platelets whereas the first phase was nearly independant of ADP. While the ability of platelet fibrinogen to aggregate platelets and to clot with thrombin were otherwise intimately interconnected, some aggregation activity remained after heat-denaturation of the platelet fibrinogen.     

Redistribution of Platelets During Discontinous Flow Platelet Pheresis

Abstract. Greater numbers of platelets are recovered during discontinuous flow centri-fugation than can be simply accounted for by the decrease in total circulating platelets in the donor. There is a linear relationship between the logarithm of the circulating platelet count and the number of plateletpheresis bowls filled. The disappearance of platelets from the peripheral circulation occurs at a greater rate in splenectomized donors than in normal donors, and the rate of platelet disappearance in normal donors is less than what would be expected if there were no in vivo platelet storage pools. The data suggest the redistribution of platelets from the spleen in normal donors during the time course of the procedure.     

Mepacrine Stains the Dense Bodies of Human Platelets and not Platelet Lysosomes

S ummary . Mepacrine, used as a vital stain, accumulates in the lysosomes of most cells. In platelets, however, it does not stain the lysosomes but is a specific marker for dense bodies.     

Platelets and the Lipoproteins: Native,Modified and Platelet Modified Lipoproteins

Under physiological conditions human blood platelets play a beneficial role in fibrinolysis and regulate the balance with prostacyclin and other factors derived from the endothelium. In response to endothelial injury, adherence of platelets to the denuded arterial surface, platelet aggregation, release of mitogens and subsequent cell proliferation characterize early fibrous plaque lesions. ‘Native’ atherogenic plasma lipoproteins which are abundant in hypercholesterolemia have been found to play a subtle role in the development of atherosclerosis. In addition, lipoproteins modulate platelet function and alter the susceptibility of platelets to different stimulating agents. The properties of ‘modified’ atherogenic lipoproteins also seem to be well documented with respect to atherogenesis. After uptake by macrophages, modified atherogenic plasma lipoproteins are thought to contribute to formation of fatty streak lesions. On the other hand, modified atherogenic lipoproteins may directly promote endothelial injury and thus favour enhanced endothelial-platelet interactions. However, the direct effects of modified atherogenic lipoproteins on platelet function have not been revealed in detail. Recent findings have documented that activated platelets themselves may promote modification of atherogenic plasma lipoproteins and thus contribute to enhanced foam cell formation. Therefore stimulation of thrombocytes, and their interaction with native and modified lipoproteins must be considered an important factor in the current concept of atherogenesis.     

ADP-Induced Aggregation of Washed Platelets Effect of Platelet and Plasma Fibrinogen

ADP-induced aggregation of washed human and porcine platelets has been studied. Plasma from a patient genetically deficient in fibrinogen lacked ADP-cofactor activity. Apyrase totally inhibited the small platelet aggregation observed after addition of fibrinogen to washed platelets. Lysates of washed porcine platelets contained 1.34 mg/10⁹ platelets of protein in the soluble form and 0.44 mg/10⁹ platelets as insoluble protein. Platelet fibrinogen in soluble fraction was 0.16 mg/10⁹ platelets. Partly purified porcine platelet fibrinogen showed cofactor activity for ADP-induced aggregation of washed porcine platelets, but compared to plasma fibrinogen a higher concentration of the platelet fibrinogen was needed to obtain the same effect.     

Platelet Density and Size: the Interpretation of Heterogeneity

S ummary . Platelets have been separated according to buoyant density using a colloidal silica-polyvinylpyrrolidone system and subjected to electronic sizing. All density populations were found to be heterogeneous in size, the most dense platelets ranging from less than 3 fl to greater than 21 fl in both man and rat. Light platelet fractions contained no platelets greater than 13 fl in either species. Cohort labelling with [⁷⁵Se]selenomethionine showed no indication of significant change in platelet buoyant density with ageing; greater specific activity found in young, dense platelets appears to be related to increased protein synthetic activity shown in vitro and likely to occur also in their precursor megakaryocytes.
It is postulated that dense, intermediate and light platelets are released synchronously by the three different ploidy classes of megakaryocyte, that varying density indicates differing structural characteristics and presumably differences in function. The present findings do not deny the possibility that platelets decrease in size with ageing but if such occurs, it is not associated with a significant change in platelet buoyant density.     

Heparin-induced Thrombocytopenia: Effect of Heparin Platelet Antibody on Platelets

S ummary . The plasma of two patients with heparin-induced thrombocytopenia has been shown to cause platelet aggregation in the presence of heparin. The platelet aggregating factor was isolated in the IgG fraction of the patients'sera suggesting that it was an antibody. This heparin anti-platelet antibody (HAP-Ab) induced platelet aggregation and release but did not cause platelet lysis, although it fixed complement. Platelet aggregation was inhibited by EDTA and by inactivation of complement. There was a significant production of malondialdehyde (MDA) and thromboxane B₂ (TXB₂) implying a role of the prostaglandin synthesis pathway in HAP-Ab induced aggregation. ADP release also appeared to be involved as apyrase blocked aggregation while hirudin, a thrombin inhibitor, had no effect. The thrombotic complications that have recently been reported in patients with heparin-induced thrombocytopenia may be explained by some effects of HAP-Ab on platelets, namely: the antibody mediated platelet factor 3 release, prostaglandin endoperoxides and thromboxane A₂ (TXA₂) production and platelet aggregation in vivo. These HAP-Ab mediated effects could be inhibited by anti-platelet drugs such as aspirin, indomethacin and dipyridamole and thus may have therapeutic implications.