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PDGF is a mitogenic protein stored in platelets and released upon platelet degranulation. Recent evidence indicates that PDGF plays an important role in both physiologic and pathophysiologic processes, particularly in tumorigenesis, wound healing, pulmonary fibrosis, and atherogenesis. In addition to its mitogenic potential, it has been reported that PDGF stimulates monocyte chemotaxis. Since the recruitment of monocytes from the peripheral vasculature is an important event in vivo, the potential role of PDGF as a monocyte chemoattractant has significant biologic implications. However, we now report that homogeneous human PDGF from platelets and a recombinant PDGF-2 homodimer do not stimulate monocyte chemotaxis. In contrast to previous reports these results indicate that PDGF is not a monocyte chemoattractant.

Summary Haemostatic plug formation in four patients with severe haemophilia A (VIII:C < 1%) was studied in skin biopsies taken at 3, 10 and 30 min and 2 h after a template bleeding time wound had been made. The primary haemostatic plug showed relatively minor changes, consisting of a delay in platelet degranulation and interdigitation. Some platelet aggregates not attached to vessels were encountered in the wound. Subsequently the primary haemostatic plug changed into a firm stable degranulated mass of interdigitated platelets. The major abnormality occurred during the fibrinous transformation. At 2 h many haemostatic plugs consisted of a thin peripheral layer of fibrin and platelet remnants around a central area containing red and white blood cells with a varying amount of plasma and only relatively few fibrin fibres. These observations suggest that fibrin formation in the periphery of the plug is less dependent of factor VIII than in central areas. The lack of fibrin formation in the centre of the plug compensating for the platelet lysis at 2 h may have caused the central erosion of the plug.

SummaryPlatelet adhesion to natural and artificial surfaces and adhesion-induced aggregation were investigated in vitro using an annular perfusion chamber. The surfaces were exposed to anticoagulated blood under identical flow conditions (~ arterial shear rates). The initial attachment of platelets (contact) appeared less surface specific than spreading and release. Fibrillar collagen was the most powerful inducer of platelet degranulation whereas elastin, microfibrils and epon were virtually inactive. Fibrillar collagen caused release also in the absence of spreading. Surface coverage with platelets did not exceed 25 % unless spreading occurred. Perfusion with platelet-free plasma or platelet-poor blood did not remove adhering platelets. However, platelets were translocated from mural thrombi to the surface by such perfusion. In addition, platelets which detached from mural thrombi adhered more readily to elastin or microfibrils than platelets from the circulating blood. The initial attachment of platelets to subendothelium was inhibited in von Willebrand’s disease, the Bernard-Soulier syndrome and at high concentrations of dipyridamole; spreading was inhibited in storage pool disease of rats, at low temperature (20° C), with EDTA (3 mM) and Prostaglandin E1 (1 μM); and adhesion-induced aggregation was inhibited in thrombasthenia, storage pool disease and after ingestion of sulfinpyrazone or Aspirin.It is concluded that the initial attachment (contact) of platelets, spreading and surface-induced release of platelet constituents are at least partially independent phenomena, the latter two being highly surface specific. At flow conditions which cause the disappearance of platelet thrombi, platelet adhesion appears as an irreversible process.

Investigations of human platelet-derived growth factor (PDGF), a potent mitogen for mesenchymal derived cells in culture [l-3], have provided a rational basis for the understanding of at least one mechanism involved in malignant transformation. PDGF is a heat-stable (loo”), cationic (isolectric point 9.8) polypeptide [4] that circulates in blood stored in the a-granules of platelets [5]. It is released from platelets into the serum during blood clotting, constituting the major polypeptide growth factor of serum. It is suggested that in uiuo PDGF is delivered during platelet degranulation at the site of injury where it participates in the process of wound healing by stimulating the proliferation and migration of connective tissue cells. We have shown recently that PDGF and the transforming protein of the simian sarcoma virus (SSV). an acute transforming retrovirus of primate origin, derive from the same or closely related cellular genes [6]. This conclusion is based on the demonstration that PDGF and the SSV transforming protein [7,8] share extensive amino acid sequence homology 16, 9, 101, have common antigenic determinants and structural conformation [ 111, and exert identical biological functions [12, 131. These findings suggest that the ability of the simian sarcoma virus to induce transformation derives from the incorporation of the PDGF gene within the retroviral genome. The resulting transforming one gene (v-sis) region within the retrovirus genome codes for a PDGF-like mitogen and is capable of inducing neoplastic transformation by the continuous production of this potent mitogen causing sustained cell proliferation. Consistent with the findings described above is the detection of v-sis-related messenger RNAs in human tumors of mesenchymal origin, such as glioblastomas, fibrosarcomas, and osteosarcomas [14]. Production of PDGF-like mitogen by these human malignant cells in culture has been reported [ 15-171. More recent studies have demonstrated that these cells synthesize, process, and release PDGF-like polvpeptides which are recognized by specific PDGFante the current understanding of PDGF structure and function; its role in the regulation of normal cell growth; and its link to malignant transformation.

Platelet-derived growth factor (PDGF) is a potent mitogen for many cultured connective tissue cells. It is present in concentrated form within the platelet alpha-granules and is believed to be released during platelet degranulation at sites of vascular injury. We have used a sensitive radioreceptor assay to measure PDGF levels in whole blood serum from normal humans [17.5 +/- 3.1 (SD) ng/mL] and baboons (2.7 +/- 1.2 ng/mL). PDGF was not detected in plasma from either species. In addition, plasma was found to substantially reduce the ability of added purified PDGF to bind to the cell surface PDGF receptor on cultured cells, suggesting that plasma may contain a PDGF-binding protein that would serve to inactivate PDGF released into plasma. Calculations of PDGF concentrations in serum have been corrected for the effects of the binding protein. 125I-PDGF injected intravenously into normal baboons was cleared rapidly from the plasma (t1/2 = two minutes). The rapid clearance of 125I-PDGF did not result from iodination damage, as purified unlabeled PDGF was cleared with comparable kinetics. The rapid clearance of purified and iodinated PDGF did not result from changes in PDGF structure during purification or from removal of PDGF-associated proteins during purification, as PDGF present in freeze-thaw lysates of fresh platelets was cleared equally rapidly. We conclude that release of PDGF at sites of vascular injury would greatly increase the local concentration of PDGF and that PDGF not localized to the site of injury would be rapidly cleared from the circulation.

Human platelet-derived transforming growth factor type beta (TGF beta) is mitogenic for fetal rat calvariae in serum-free organ culture. It enhances DNA synthesis in short (24-h) and long (48- to 96-h) term cultures, but produces no significant stimulatory effects on bone collagen synthesis or alkaline phosphatase activity (two parameters of differentiated osteoblastic cell-type function) when present continuously in culture. Transitory treatment with TGF beta, however, induces a subsequent stimulation of collagen and noncollagen protein synthesis that depends on prior cell replication, suggesting an increase in the number of newly differentiated bone cells. In addition, TGF beta increases prostaglandin release, but this effect is probably unrelated to its mitogenic function. TGF beta activity is also found in culture medium conditioned by fetal rat calvariae, and the bone-derived factor produces effects similar to those of the human platelet factor. This polypeptide, therefore, may have an important function in early stages of bone development as well as bone repair after trauma-induced platelet degranulation.

The pathogenesis of atherosclerosis is hypothesized to occur as a response to various forms of injury to the lining arterial endothelial cells. The resulting endothelial alterations could potentially lead to interactions between platelets in the circulation and the underlying subendothelial connective tissue or with the altered endothelial cells themselves. Such interactions provide an opportunity for platelet degranulation and release of a platelet-derived growth factor. This factor has been shown in cell culture to be an extremely potent mitogen and will induce DNA synthesis and cell multiplication of a number of cells including smooth muscle cells, fibroblasts, and other mesenchymally derived cells. Chronic endothelial injury and repeated interactions between platelet-derived mitogens, plasma components, and the underlying arterial smooth muslce cells would promote the progression of the intimal proliferative lesions of atherosclerosis that lead to the clinical sequelae associated with this disease process.

A specific radioimmunoassay for type beta transforming growth factor (TGF-beta) was developed and used to show that human platelets treated with thrombin release TGF-beta as a consequence of degranulation. The thrombin concentrations required to induce release of TGF-beta parallel those concentrations that release the alpha-granule marker, beta-thromboglobulin. Related studies showed that TGF-beta acts on early passage, explant cultures of bovine aortic smooth muscle cells by inhibiting the effect of mitogens on proliferation of subconfluent cell monolayers yet synergizing with mitogens to stimulate growth of the same cells when cultured in soft agar. The results show that primary cultures of bovine aortic smooth muscle cells and established normal rat kidney cells behave similarly with regard to TGF-beta action. Moreover, the data suggest that platelet-mediated proliferation of aortic smooth muscle cells in vivo may not result solely from the stimulatory effect of platelet-derived growth factor (PDGF), but rather from an interaction of platelet factors which has the intrinsic ability to limit as well as stimulate mitosis.

Imidazole and compound L8027 (selective inhibitors of thromboxane synthase) produced parallel inhibition of malonaldehyde and thromboxane B2 secretion induced by collagen or thrombin in gel-filtered suspensions of human platelets. Comparing the effects of these inhibitors and aspirin on secretion of granule constituents indicated that platelet degranulation depends mainly on thromboxane production; prostaglandin endoperoxides contributed little.

Subcellular fractionation of human platelets labeled with radioactive sodium chromate was utilized to study distribution and characterization of the 51Cr binding sites in these cells. The cytoplasmic fraction of the platelets contained the major portion of the radioactivity while microsomal and mitochondrial fractions played a minor role in the binding of chromate. The stromal sediment had approximately one third of the total radioactivity. Only about 20 per cent of the 51Cr taken up by the platelets was in a free ionic form and was in the cytoplasmic fraction. The major portion of the 51Cr in the platelets was bound to nucleotides in the platelet cytoplasm. Nucleotides of the α-granules had no radioactive chromate. Hypertonic media and platelet antiserum produced marked release of 51Cr from the platelets, whereas aggregating agents produced none. In physiologic conditions, elution of 51Cr from labeled platelets is very limited because there is only little 51Cr in free ionic form in the cells. Release of considerable amount of 51Cr from the platelets can occur only in case of cell damage with release of nucleotides from the cytoplasmic pool. This was seen with hypertonic solutions and with platelet antibody. Platelet degranulation, as it occurs with aggregating agents, is not associated with release of 51Cr since the granular pool of nucleotides is not labeled by this compound.

Summary. The lowest concentration of ADP and adrenaline which resulted in a secondary wave of platelet aggregation at 37°C was determined in 60 normal subjects. The range of such ‘threshold’ concentrations was 0.2–1.4μM for ADP and 0.1–1.0μM for adrenaline. The mean threshold concentration of each reagent was unrelated to age but was higher for men than for women; this difference disappeared when the results were corrected for the effect of differences in PCV on the citrate concentration in platelet-rich plasma. The variation between separate determinations of threshold concentration in the same individual was of the same order as that between individuals; no evidence was found that the threshold concentration of either reagent was related to the phase of the menstrual cycle in four young women tested repeatedly. There was a highly significant correlation between the threshold concentrations of the two reagents in individual subjects. No correlation was found between the threshold concentration of either reagent and the total platelet ATP, ADP or ATP: ADP ratio. Concentrations of adrenaline at or slightly above the threshold caused the release of up to about 50% of the platelet ADP and 25% of the ATP during the second phase of aggregation, while lower concentrations produced no significant release. The ultrastructural changes during the first phase of aggregation were similar with all concentrations of ADP tested; threshold concentrations and above caused dense packing of platelets during the second phase, with the formation of giant pseudopodia and no tendency to disaggregation during the period of observation, while at lower concentrations disaggregation was accompanied by a retraction of pseudopodia. There was no clear evidence of platelet degranulation during either first or second phase aggregation.

Electron microscopy has confirmed previous studies and has provided much new information on the mechanism of endotoxin-platelet interaction. The Boivin lipopolysaccharide preparation is particulate, and on electron microscope examination appears as a three-layered structure, morphologically similar to bacterial cell wall. In vitro and in vivo experiments have demonstrated that these endotoxin particles adhere to platelets. In some species, particularly the rabbit, this is associated with loss of platelet contents, due to lysosomal and cell membrane lysis, resulting in platelet sphering and apparent cell death. Serial observation of degranulating platelets and metabolic studies indicate that although some platelet engulfment of endotoxin occurs, degranulation is not dependent upon phagocytosis. Several observations suggest that these endotoxin effects are mediated through immune mechanisms: (1) Inactivation of complement in the suspending plasma by heating to 56°C, anticoagulation with EDTA, a reaction temperature of 5°C, ammonium hydroxide incubation, and adsorption with either zymosan or washed antigen-antibody complexes, inhibits both endotoxin adherence and platelet degranulation. (2) The reaction requires a plasma cofactor, possibly antibody, which can be adsorbed out by endotoxin. (3) Endotoxin adheres selectively to nonprimate platelets and primate red cells, a pattern conforming to immune adherence, a phenomenon requiring antigen, antibody, and complement. It is suggested that endotoxin-induced platelet damage is dependent upon the intimate contact provided by immune adherence. We have not established whether degranulation is an endotoxin or complement effect. The species variation in susceptibility to endotoxin also merits further investigation.

Thrombocytopenia is a consistent finding and one of the earliest hematological abnormalities in horses acutely infected with equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus. Multifactorial mechanisms, including immune-mediated platelet destruction and impaired platelet production, are implicated in the pathogenesis of EIAV-associated thrombocytopenia. This study was undertaken to investigate whether regenerative thrombopoiesis and platelet destruction occurred in ponies acutely infected with EIAV. Circulating large, immature platelets were increased in ponies acutely infected with EIAV late in the infection when platelet count was at a nadir. Morphometric analysis of bone marrow from acutely infected ponies revealed significant increased in megakaryocyte area and megakaryocyte nuclear area. A trend toward increased numbers of megakaryocytes was also observed. Platelets from acutely infected ponies had increased surface-bound fibrinogen and ultrastructural changes consistent with in vivo platelet activation. Platelets also had hypofunctional aggregation responses to three agonists in vitro. We conclude that thrombocytopenia in ponies acutely infected with EIAV is regenerative and suggest that bone marrow platelet production is not severely compromised in these ponies. Our findings reveal that in vivo platelet activation occurs in ponies acutely infected with EIAV, and as a result platelets are hypofunctional in vitro. Activation of platelets in vivo may cause platelet degranulation or formation of platelet aggregates, which would result in removal of these damages platelets from circulation. This may represent a form of nonimmune-mediated platelet destruction in ponies acutely infected with EIAV.

The ultrastructural changes and the kinetics of 14C-serotonin release during the adhesion-aggregation reaction of human platelets induced by Thrombofax® (Ortho Pharmaceuticals), a commercial cephalin prepared as an ether extract of acetone dried bovine brain, were analyzed. The initial phase of interaction of Thrombofax with platelets is characterized by the development of adhesion sites on normally shaped cells; thereafter, loss of discoid shape, pseudopod formation and inward movement of granules, surrounded by a circumferential bundle of microtubules, become apparent. Measurements of 14C-serotonin release show that these ultrastructural changes precede the release reaction. The later stages which are detectable turbidometrically and which only occur in stirred samples and in the absence of release inhibitors, are characterized mainly by large aggregate formation, platelet degranulation and strong release of 14-C-serotonin.

Plasma beta-thromboglobulin (beta-TG), a platelet-specific protein, is a marker of intravascular platelet degranulation. We measured plasma beta-thromboglobulin by radioimmunoassay in 13 patients with thrombocytopenia of various etiologies to determine whether or not the test is clinically useful in the differential diagnosis of thrombocytopenia. Four patients with intravascular platelet consumption (three with thrombotic thrombocytopenic purpura and one with vasculitis) had significantly higher plasma beta-thromboglobulin levels than four patients with extravascular platelet destruction due to idiopathic thrombocytopenic purpura. Five patients with thrombocytopenia and decreased numbers of megakaryocytes in the bone marrow also had beta-thromboglobulin levels that were not elevated. Two patients with thrombotic thrombocytopenic purpura achieved clinical remission associated with a decrease in beta-TG level to the normal range. Plasma beta-thromboglobulin determination can be useful in determining the mechanism of thrombocytopenia when bone marrow examination demonstrates adequate megakaryocyte numbers.

The experiments presented here were undertaken from the point of view that the reaction sequence ATP ⇒ ADP ⇒ AMP ⇒ adenosine, occurring extracellularly, is a key sequence contributing to regulation of the time course of cell and tissue response during crisis or signaling events. Adenine nucleotides are released into the pericellular space during platelet thrombus formation, neurotransmission, catecholamine release from the adrenal medulla, strenuous exercise, and shock. Since the extent of phosphorylation can profoundly modify the physiological effects of extracellular adenine nucleotides, it is important to discover how the time course of their hydrolysis after release is controlled. Plasma membrane 5′-nucleotidase is known to be inhibited by ATP and ADP. This property suggests the possibility of a particularly interesting feature of the regulation of the time course of the extracellular conversion of ATP or ADP to adenosine. When the extracellular concentration of ATP or ADP is low, it should be quickly degraded all the way to adenosine. When a bolus of ATP or ADP of high enough concentration to inhibit 5′-nucleotidase appears, the rate of adenosine appearance should be diminished. We have examined the time course of this reaction sequence during recirculation of substrate solutions over cultured pig aortic endothelial cells attached to polystyrene beads. This arrangement permits study of the reactions in incubations with volume-to-cell-surface ratios approaching those of small blood vessels. Reaction rates are proportional to cell number, and the kinetics of the ectoenzymes are reproducible for at least 8 hr. When endothelial cells are presented with an initial bolus of ATP, high concentrations of the intermediates ADP and AMP develop before significant conversion of AMP to adenosine occurs. Further, there is a paradoxical dependence of the initial rate of adenosine appearance on the initial ATP concentration: the higher the initial ATP concentration, the slower is the conversion of AMP to adenosine during early time points. The behavior of the system was modeled assuming one enzyme for each nucleotidase reaction and testing for inhibition of each reaction by its product and for inhibition of 5′-nucleotidase by ATP and/or ADP. Kinetic constants for each reaction were estimated by fitting simulated reaction curves to observed time courses. K MS estimated in this way agreed well with those obtained from initial velocity measurements: for the ATPase, 300 μM; the ADPase, 240 μM; and 5′-nucleotidase, 26 μM. Maximum velocities varied from cell batch to cell batch but stayed in approximately the proportion ATPase : ADPase : AMPase = 6 : 1.5 : 1. The data for the rate of appearance of adenosine can only be fit if the model incorporates inhibition of 5′-nucleotidase by ATP or ADP. The KI for ADP is 5 μM. Product inhibition does not contribute significantly to the regulation of the pathway. These kinetic properties tend to maximize the time separation of the intermediate pools. In vivo, at sites of platelet degranulation, they would tend to create a time gap proportional to the size of the initial release between release of ADP (a proaggregatory milieu) and the appearance of adenosine (an antiaggregatory milieu).

A protein layer coats the dialysis membrane on the first contact with blood: the nature of this layer may influence the following biological, immunological and clinical events such as intrinsic coagulation system and complement cascade activation (1). The nature of adsorbed protein on dialysis filters has been studied by the use of SDS-PAGE following various desorption procedures. Unfortunately results seem not easily reproducible among different studies: protein-surface interaction depends upon membrane chemical structure and varies among in vitro experiments and in clinical use. In order to better characterize the nature of the protein layer we have developed a new immunohistochemical method with the fluorescent antibody technique already used for the kidney biopsy. Biocompatibility of four different dialysis membranes was evaluated comparing protein cake deposition, plasma anaphilatoxin generation an platelet degranulation as B-TG release.

The morphological changes occurring in xenograft hyperacute rejection, using guinea-pig to rat cardiac transplants, have been studied. Ultrastructural evidence of platelet aggregation in myocardial capillaries and veins first occurred two minutes after revascularisation. Small amounts of fibrin, associated with platelet degranulation occurred at three minutes. From four minutes, platelet aggregates increased in number and endothelial damage first appeared in those vessels containing platelet aggregates. Rejection was complete by 12 minutes and platelet aggregates then filled many capillaries, veins, arterioles and arteries. Polymorphonuclear leucocyte infiltration was not seen.

Chikungunya virus infectivity was markedly stabilized in the presence of washed suspensions of human platelets but rapidly disappeared in similar preparations of rabbit platelets. Supernatant fluids collected from human platelets had some stabilizing effect on chikungunya virus over a 6-day incubation period at 37 °C. Rabbit platelet supernatant fluid had no virus-stabilizing effect, nor did it demonstrate any capacity to inactivate virus as compared to whole rabbit platelet preparations. Thin-section electron microscopy demonstrated that chikungunya virus formed an association with human platelets by becoming entrapped in platelet aggregates; during this process some of the platelets appeared to have undergone degranulation and lysis. Rabbit platelets exposed to chikungunya virus for 24 h demonstrated a considerable amount of platelet degranulation and lysis but virus was not visualized eitherin association with platelet membranes or within phagocytic vacuoles in the platelet cytoplasm. Human platelets, which appear to be more stable under these incubation conditions, may protect chikungunya virus infectivity from heat inactivation by surrounding viruses with large platelet aggregates whereas rabbit platelets, which appear to be more fragile, do not afford this type of protection. Thus, chikungunya virus in the presence of rabbit platelets may become inactivated by heat or may become bound irreversibly to membranes in such a fashion that infectivity assay and electron microscopy techniques may prove to be too insensitive for detection of virus.

Type β-transforming growth factor (TGF-β) is a low-molecular-weight protein with a broad array of biologic effects, including the ability both to stimulate and inhibit the proliferation of cells in culture (Tucker et al., 1984; Roberts et al., 1985; Sporn et al., 1986). This chapter focuses on a single aspect of TGF-β action: its ability to induce a striking wound-healing response in several whole-animal and cell-culture model systems. These results, considered with experiments showing storage of TGF-β in platelets and release of TGF-β during platelet degranulation, strongly suggest that this growth factor has an important physiologic role as an endogenous mediator of wound repair and fibrosis.

Macrophage cholesterol accumulation is an early event in atherogenesis. Platelet secretory products have the potential to affect macrophage cholesterol accumulation through their effect on cellular lipoprotein uptake via the low density lipoprotein (LDL) or the scavenger receptor pathways. Preincubation of human monocyte-derived macrophages (HMDM) for 16 hours at 37°C with serotonin, ADP, fibrinogen, fibronectin and platelet-derived growth factor (PDGF), followed by washout of these substances, significantly enhanced LDL uptake by 25% to 75%, whereas acetyl LDL (AcLDL) degradation (AcLDL is taken up by the scavenger receptor), was substantially reduced by 40% to 60% (except for ADP). The effect of serotonin (0 to 75 μmol/L) on macrophage interaction with lipoproteins was further analyzed and revealed a dosedependent effect on both stimulation of macrophage LDL degradation and cholesterol esterification by up to 2.5 times, as well as an inhibition of the cellular uptake of AcLDL by up to 1.5 times. Analysis of the regulatory effect of serotonin on macrophage lipoprotein uptake revealed that the main effect of serotonin on the uptake of both lipoproteins was to change the affinity of the lipoproteins toward their specific receptor without a significant effect on the number of binding sites. The next questions addressed are whether substances that are known to be secreted by activated platelets can also modify LDL, and whether this modification can alter the interaction between LDL and macrophages. LDL treated with all of the studied substances demonstrated significantly enhanced cellular degradation compared with untreated LDL. The data thus demonstrate that substances such as those released from activated platelets can selectively affect macrophage LDL and scavenger receptor activities. Thus, platelet secretory products released during platelet degranulation can possibly modulate arterial macrophage cholesterol metabolism and can affect the atherosclerotic process.

The intravenous injection of the mast cell degranulator C 48/80 (1 mg/kg) in rats did not produce thrombocytopenia nor circulating platelet aggregates but sensitized the platelets to aggregate upon turbulence challenge. Such turbulence-induced platelet aggregation was not accompanied by formation of thromboxane B2. Electron microscopy revealed absence of platelet degranulation. Turbulence-induced platelet aggregation was completely prevented by pre-treatment of the rats with cyproheptadine, dipyridamole and VK 774, partially with ketanserin (5HT2-receptor antagonist), but not with methysergide (anti-serotonergic drug), pyrilamine (antihistaminic drug), suprofen, aspirin (cyclo-oxygenase inhibitors), phentolamine, propranolol, flunarizine, lidoflazine, oxycoumarin or Trasylol®. Combined treatment with the anti-histaminic drug pyrilamine and the 5HT2-receptor antagonist ketanserin resulted in a dose-related inhibition for ketanserin of the turbulence-induced platelet aggregation. These experiments point to an interaction between histamine and 5-hydroxytryptamine in the platelet activation by mast cell released mediators.

The membranolytic effects of microcrystalline monosodium urate (MSU) on biologic membranes are well documented for a number of cells and subcellular structures [1–3]. This process has been ascribed to hydrogen-bond formation between the crystal and membrane phospholipids and is blocked by strong hydrogen acceptors such as proteins or anionic polymers [1]. Since the acute inflammatory response of gouty arthritis occurs in protein-rich joint fluid, we have examined the protein adsorptive properties of MSU crystals. The effects of adsorbed protein on polymorphonuclear leukocytes and platelet degranulation were studied and are reported in detai1.

Compound 48/80 degranulates mast cells and release histamine and serotonin. Since platelet degranulation is an important aspect of platelet function, the effect of this compound on platelets and platelet aggregation were investigated by transmission and scanning electron microscopy. Low concentrations of 48/80 did not alter platelet ultrastructure but high concentrations suppressed platelet pseudopod formation and altered the surface connecting system. Low concentrations of 48/80 interfered with platelet aggregation by thrombin and collagen but not by ADP; platelets incubated with high concentrations of 48/80 did not aggregate when exposed to ADP or collagen but the addition of large quantities of thrombin overcame this inhibition and caused platelet aggregation as well as plasma clotting. The effect of thrombin on platelets exposed to 48/80 was not related to the presence of fibrin. The observations provide evidence that the platelet membrane plays an essential role in aggregation and that the mechanisms leading to platelet release and aggregation by various agents may be different.

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 – 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2’,7’-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a coagulation factor XIII (F13A1) 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin–α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Low molecular weight heparins are widely used in the prophylaxis and treatment of thrombotic disorders. The effect of low molecular weight heparins on coagulation was examined ultrastructurally in an animal model. A test and a control group was formed, each consisting of five rabbits. Nadroparine (225 Institute of Chaoy Unit/kg twice daily) was applied to the test group for 10 days. The control group received 1 ml saline solution subcutaneously. Blood and vascular tissue samples collected at the end of the 10th day were evaluated under a JEM 100 B electron microscope. Platelet degranulation and agglutination was observed in the control group. Fibrin materials were detected in the cytoplasms and surroundings of degranulated platelets. Erythrocyte accumulation was remarkable on the vascular endothelium with intact coagulation periods. In the test group, outer membranes of platelets, hyalomere, and granular structures in the granulomeres were detected to be nearly intact. There were rare erythrocytes in the large vascular lumens. The aggregation phase had occurred but no agglutination was detected. Nadroparine seems to preserve consistency of lipoprotein membranes of platelets and granular structures containing enzymes, which contribute to the coagulation mechanisms.

On stimulation by strong agonists, platelets release the contents of 3 storage compartments in 2 apparent waves of exocytosis. The first wave is the release of α- and dense core granule contents and the second is the release of lysosomal contents. Using a streptolysin O-permeabilized platelet exocytosis assay, we show that hexosaminidase release is stimulated by either Ca++ or by GTP-γ-S. This release step retains the same temporal separation from serotonin release as seen in intact platelets. This assay system was also used to dissect the molecular mechanisms of lysosome exocytosis. Lysosome release requires adenosine triphosphate and the general membrane fusion protein, N-ethylmaleimide sensitive factor. Uniquely, 2 syntaxin t-SNAREs, syntaxin 2 and 4, which localize to granules and open canalicular membranes, together with the general target membrane SNAP receptor (t-SNARE) protein SNAP-23 appear to make up the heterodimeric t-SNAREs required for lysosome exocytosis. These studies further show that regardless of stimuli (Ca++or GTP-γ-S) serotonin and hexosaminidase release requires the same membrane fusion machinery.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed "reperfusion injury." Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.

• Levels of the platelet degranulation product β-thromboglobulin (BTG) and the fibrinogen degradation product fibrinopeptide A (FPA) were measured in 26 asymptomatic subjects (group 1), 17 patients with peripheral vascular disease (PVD) (group 2), and 12 patients with PVD and bypass grafts (group 3). Mean BTG and FPA levels were elevated in both groups 2 and 3, indicating increased thrombotic activity in patients with PVD. Results of serial BTG and FPA assays in group 3 patients suggested a trend downward. These markers may be useful for estimating disease severity and prognosis, extent of graft healing and patency, and efficacy of therapeutic intervention. ( Arch Surg 1987;122:897-900)