Absence Of Platelets Research Articles

Adhesion of Polymorphonuclear Leukocytes to Protein-Coated and Platelet Adherent Surfaces

Since protein adsorption and platelet adhesion are likely to precede significant contact of leukocytes with the surfaces of artificial organs, we have chosen to study polymorphonuclear leukocyte (PMN) adhesion in a sequential manner. The work presented here deals with the effects of flow and surface type on PMN adhesion to fibrinogen- and albumin-coated glass. We compared direct adhesion to adsorbed protein with adhesion to adsorbed protein having adherent platelets. These experiments were designed to see if PMN's might preferentially adhere to albumin or fibrinogen and whether a particular morphological form of adherent platelet could promote PMN adhesion. The adhesion of PMN's to spread platelets on albumin or fibrinogen occurs to a greater extent than in the absence of platelets. Adhesion of PMN's to spread platelets may be an important mechanism for their depletion from the circulation during artificial organ use.

The influence of thrombin and platelets on fibrin clot lysis rates in vitro: a study using a clot lysis system consisting of purified human proteins

An in vitro clot lysis model system was developed in order to study the effects of several components on fibrin formation and fibrin degradation. The system consisted of the following human proteins: ( 125I)-fibrinogen, plasminogen, F XIII, t-PA, PAI, α 2-antiplasmin and platelets. Clot formation was induced with α-thrombin in the presence of Ca 2+-ions. Increasing amounts of tissue-type plasminogen activator or plasminogen induced an acceleration of clot lysis, without affecting the clotting time. Prolonged clot lysis times were observed when increasing concentrations of fibrinogen, plasminogen activator inhibitor (PAI) or α-antiplasmin ( α 2-AP) were added, without affecting the clotting time. F XIII (in the absence of α 2-AP) and phospholipid did not influence clot lysis times or clotting times. However, in the presence of α 2-AP, F XIII induced a significant prolongation of clot lysis times. The characteristics of this clot lysis system agree with what is known from previously performed fibrinolytic studies. The addition of platelets to the system resulted in a significant decrease in clot lysis rate. At higher α-thrombin concentrations the inhibitory effect of platelets was more pronounced. α-thrombin, in the absence of platelets, did not influence lysis rates. When Reptilase ® was used instead of α-thrombin to induce fibrin formation, platelets had no effect on clot lysis, suggesting that the α-thrombin induced platelet release reaction is responsible for the inhibition of clot lysis. Release of PAI from platelets was found to be dependent on the α-thrombin concentration used and is proposed as the explanation for the finding that in the presence of platelets clot lysis is inhibited by α-thrombin in a dose-dependent way.

Role of platelets in contraction of canine tracheal muscle elicited by PAF in vitro.

To elucidate mechanisms of platelet-activating factor (PAF)-induced contraction, we studied the effect of PAF on 203 canine tracheal smooth muscle (TSM) strips from 45 dogs in vitro in the presence and absence of platelets. PAF (10(-11) to 10(-7) M) alone caused no contraction of TSM even in the presence of airway epithelium. In the presence of 2 x 10(5) platelets/microliter, PAF was an extremely potent contractile agonist (threshold 10(-11) M). This response was inhibited by the PAF antagonist, CV-3988 (10(-6) M), and reversed by the serotonin antagonist, methysergide (EC50 = 3.7 +/- 0.79 x 10(-9) M). Neither atropine nor chlorpheniramine (10(-9) to 10(-6) M) attenuated the response to PAF + platelets. In the presence of platelets, 10(-7) M PAF caused an increase in perfusate concentration of serotonin from 0.93 +/- 0.037 x 10(-8) to 1.7 +/- 0.046 x 10(-8) M (P less than 0.001). Tachyphylaxis, previously demonstrated to be irreversible, was shown to be a platelet-dependent phenomenon; contraction could be repeated in the same TSM after addition of fresh platelets. We demonstrate that PAF-induced contraction of canine TSM is caused by the release of cellular intermediates such as serotonin from platelets. We also demonstrate the site of PAF-induced tachyphylaxis in airway smooth muscle contraction.

Platelet-enhanced apolipoprotein E production by human macrophages: a possible role in atherosclerosis

Cholesterol-loaded human monocyte-derived macrophages increase their production of apolipoprotein E (apoE). Although cholesterol loading is often achieved with modified plasma lipoproteins, macrophages can be loaded also by coculture with platelets. Therefore, the relationship between platelet-mediated cholesteryl ester accumulation and apoE secretion was examined. Macrophages were isolated by adherence and cultured for 6 days in serum-free medium. Secreted apoE was measured with a sensitive solid-phase radioimmunoassay. Maximum apoE secretion by the adherent macrophages from 5 x 10(6) peripheral blood mononuclear cells was obtained with 3 x 10(8) platelets and was ten-fold greater than control cells cultured in the absence of platelets. Platelet-mediated apoE secretion was consistently greater than that obtained by culture with either native or acetylated low density lipoproteins. Whereas the 1000 g supernatants of unstimulated platelets were poor inducers of apoE secretion, substances rich in cholesterol that were shed from thrombin-stimulated platelets and recovered in the 1000 g supernatants were almost as active as intact platelets. In all studies, platelet-induced secretion of apoE paralleled the capacity of platelets to induce macrophage cholesterol accumulation, indicating that macrophage apoE secretion was readily influenced by macrophage cholesterol metabolism.

Macrophage eicosanoid formation is stimulated by platelet arachidonic acid and prostaglandin endoperoxide transfer

The present study was designed to determine whether platelets transfer arachidonic acid or prostaglandin endoperoxide intermediates to macrophages which may be further metabolized into cyclooxygenase products. Adherent peritoneal macrophages were prepared from rats fed either a control diet or an essential fatty acid-deficient diet, and incubated with a suspension of washed rat platelets. Macrophage cyclooxygenase metabolism was inhibited by aspirin. In the presence of a thromboxane synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, immunoreactive 6-ketoprostaglandin F 1 α formation was significantly increased 3-fold. Since this increase was greater (P <00.01) than that seen with either 7-(1-imidazolyl)hepatanoic acid-treated platelets or aspirin-treated macrophages alone, these results indicate that shunting of endoperoxide from platelets to macrophages may have occurred. In further experiments, macrophages from essential fatty acid-deficient rats were substituted for normal macrophages. Essential fatty acid-deficient macrophages, depleted of arachidonic acid, produced only 2% of the amount of eicosanoids compared to macrophages from control rats. When platelets were exposed to aspirin, stimulated with thrombin, and added to essential fatty acid-deficient macrophages, significantly more immunoreactive 6-ketoprostaglandin F 1 α was formed than in the absence of platelets. This increased macrophage immunoreactive 6-ketoprostaglandin F α synthesis, therefore, must have occurred from platelet-derived arachidonic acid. These data indicate that in vitro, in the presence of an inhibition of thromboxane synthetase, prostaglandin endoperoxides, as well as arachidonic acid, may be transferred between these two cell types.

Intrinsic versus extrinsic coagulation. Kinetic considerations.

A study to compare the kinetics of activation of factor IX by Factor XIa/Ca2+ and by Factor VIIa/tissue factor/Ca2+ has been undertaken. When purified human proteins, detergent-extracted brain tissue factor and tritiated-activation-peptide-release assays were utilized, the kinetic constants obtained were: Km = 310 nM, kcat. = 25 min-1 for Factor XIa and Km = 210 nM, kcat. = 15 min-1 for Factor VIIa. The kinetic constants for the activation of Factor X by Factor VIIa/brain tissue factor were: Km = 205 nM, kcat. = 70 min-1. Predicted rates for the generation of Factor IXa and Factor Xa were obtained when human monocytic tumour U937 cells (source of tissue factor) and Factor VIIa were used to form the activator. In other experiments, inclusion of high-Mr kininogen did not increase the activation rates of Factor IX by Factor XIa in the presence or absence of platelets and/or denuded rabbit aorta. These kinetic data strongly indicate that both Factor XIa and Factor VIIa play physiologically significant roles in the activation of Factor IX.

A platelet-derived immunoregulatory serum factor with T cell affinity.

A factor in mouse and human serum which enhances immune responses to SRBC and TNP-Ficoll in mice has been described. This factor completely reverses immunosuppression induced by syngeneic lymphoma (RCS) or allogeneic lymph node cells. T lymphocytes bind this factor, since the activity can be removed from serum by absorption with T cell containing spleen cells or cloned cytotoxic T lymphocytes. Furthermore, the factor is active only in the presence of mature T cells. Platelet alpha-granules seem to be the source of this serum factor because 1) the activity is lacking in plasma, in serum prepared in the absence of platelets, and in serum prepared from a patient lacking platelet alpha-granules and 2) isolated mouse or human platelets release an immunostimulatory factor with very similar properties upon incubation with thrombin.

Modulation of Pokeweed Mitogen‐Induced Human B‐Cell Differentiation by Aggregated IgG

The modulatory effect of human heat-aggregated IgG on human B-cell differentiation induced by pokeweed mitogen was investigated with three experimental protocols. Pulse exposure to aggregated IgG, followed by extensive washings before culture, of peripheral blood mononuclear cell suspensions rigorously depleted of platelets and containing less than 4% monocytes resulted in a selective decrease of the numbers of IgG-containing cells and IgG-secreting cells, whereas a simultaneous decrease of the numbers of cells producing IgG and, to a lesser extent, of those producing IgM or IgA was observed when the pulsed suspensions contained platelets and more than 4% monocytes. This non-isotype-specific suppression was shown to be more pronounced when aggregated IgG and platelets were present in the cell suspensions throughout the cultures. The results suggest that two distinct suppressor pathways can be triggered by aggregated IgG. The first one is restricted to cells producing the matching isotype, in the absence of platelets, with few monocytes in the cell suspensions. The second one leads to a nonspecific suppression of the three major Ig classes. It requires the presence of platelets and/or a high percentage of monocytes and, although it remains to be demonstrated, is probably mediated by prostaglandin E2.

Activation of factor IX bound to cultured bovine aortic endothelial cells.

Previous studies have shown that factor IX and its activated form, factor IXa, bind to cultured vascular endothelial cells and that cell-bound factor IXa retains its procoagulant activity. The present studies provide evidence that factor IX bound to cultured bovine aortic endothelial cells can be activated. Factor IX activation was assessed by finding cleavage of the factor IX molecule on NaDodSO4/polyacrylamide gel electrophoresis and by the generation of procoagulant activity as assessed by thrombin-treated factor VIII-dependent generation of factor Xa activity. Cell-bound factor IX (0.8 micrograms per 4 X 10(8) cells per ml) could be activated by factor XIa (5 micrograms/ml) or by factor VIIa (0.1 micrograms/ml) without exogenous tissue factor when endothelial cells were treated with phorbol ester and acquired tissue factor-like procoagulant activity. Regardless of how factor IX was activated, the cell-bound factor IXa required thrombin-treated factor VIII and calcium, but not exogenous phospholipid, to activate factor X. In further experiments, factor X bound to endothelial cells specifically and reversibly with a dependence on calcium and with a lower affinity (half-maximal at 480 nM) than factor IX. At saturation, 9.1 X 10(6) factor X molecules were bound per cell. After activation of factor X by factor IXa, approximately 50% of the factor Xa formed could be eluted from the cells by 10 mM EDTA, suggesting that the factor Xa was cell associated. These observations indicate that endothelial cells can bind and promote the activation of factors IX and X in the absence of platelets or exogenous phospholipid.

Improved functional recovery of the isolated rat heart after 24 hours of hypothermic arrest with a stable prostacyclin analogue (ZK 36 374)

Prostacyclin (PGI 2) can protect the heart against ischemia, i.e. it can reduce myocardial damage [9, 10]. PGI 2 protects the myocardium in vivo by preventing platelets from clumping and by dispersing preformed platelet aggregates [1, 14]. However, also in the absence of platelets, PGI 2 was shown to protect the myocardium against ischemia at concentrations that did not affect smooth muscle tone in the vessel wall [2]. This protective effect of PGI 2 in vitro might be related to a stabilization of cell membranes in adrenergic nerve endings and hence to the prevention of ischemia-induced catecholamine release [13]. The instability of PGI 2, both in vitro and in vivo, limits its application during long ischemic periods. Recently, a stable prostacyclin analogue, ZK 36 374, was demonstrated to have several prostacyclin-mimetic activities, both in vitro and in vivo [11, 12]. In this communication we report upon the beneficial effect of this stable prostacyclin analogue at a low concentration (4 n m) on the extent of ischemic damage, on the recovery of myocardial function and on the occurrence of arrhythmias in the isolated rat heart after 24 h hypothermic cardiac arrest.

The Relation of Blood Platelets to Hemorrhagic Disease

It is my purpose in this paper to report three cases and experiments which furnish additional evidence to show that the blood platelets play a part in stopping hemorrhage, and that one type of hemorrhagic disease may be attributed to an extreme reduction in the number of platelets. The cases possibly explain the relief which sometimes follows transfusion in hemorrhagic disease. It is my purpose also to describe a method for studying hemorrhage called the bleeding time, and to describe briefly a simple method for determining the coagulation time. In the cases there was marked hemorrhagic diathesis, a normal coagulation time, and almost an absence of platelets. Transfusion was performed in each case. After transfusion there was a marked increase in the number of platelets and remarkable relief of hemorrhage. When the platelet counts returned to their previous low level, hemorrhages returned. Later in the course of the disease in

Der Einfluß von Blutfaktoren auf den Myosingehalt kultivierter Gefäßmuskelzellen

Heparin, sodium butyrate, and the absence of platelet derived factors in the nutrient medium are able to inhibit the decrease of the content of myosin in cultivated arterial smooth muscle cells. Factor VIII and thrombin have no significant influence on this process.

Multimeric structure of platelet factor VIII/von Willebrand factor: the presence of larger multimers and their reassociation with thrombin- stimulated platelets

The multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

Multimeric Structure of Platelet Factor VIII/von Willebrand Factor: The Presence of Larger Multimers and Their Reassociation With Thrombin-Stimulated Platelets

AbstractThe multimeric structure of platelet factor VIII/von Willebrand factor (FVIII/vWF) in cell extracts and in collagen and thrombin releasates has been analyzed by SDS polyacrylamide gel electrophoresis followed by detection with 125I-anti-FVIII/vWF. Platelets contained larger multimers than those normally present in plasma. When secreted FVIII/vWF was analyzed, all platelet forms were released from collagen-stimulated platelets. In contrast, in thrombin releasates the larger multimers were lost in a manner dependent on divalent cations, time, and thrombin dose. This loss could not be accounted for by modification of FVIII/vWF by thrombin or platelet enzymes since no effect of thrombin on the multimeric structure of FVIII/vWF in the absence of platelets or in the presence of platelet lysates was observed. Large multimers of 125I-labeled purified FVIII/vWF underwent divalent cation-dependent association with platelets in the presence of thrombin, indicating that the loss of FVIII/vWF from thrombin releasates was due to reassociation with the platelet. These studies show a structural difference between platelet and plasma FVIII/vWF that suggests a specific role for platelet FVIII/vWF in hemostasis.

Contributions of Human Platelets to the Proteolytic Activation of Blood Coagulation Factors XII and XI

Previous studies have suggested that human platelets can promote the activation of factor XI by two different mechanisms, one requiring factor XII and ADP-treated platelets and the other requiring collagen-treated platelets in the apparent absence of factor XII. To investigate these hypotheses, isolated platelets were tested for their capacity to promote the activation and cleavage of purified factors XII and XI in various mixtures of purified factor XII, kallikrein, high molecular weight (HMW) kininogen, and factor XI. The activation of factor XII or factor XI was tested in clotting assays using plasmas deficient in factor XII, XI, IX, or VII and the limited proteolytic cleavage of 125l-labeled factor XII or XI was assessed using sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of reducing agents. That ADP- or collagen-treated platelets can promote the proteolytic activation of factor XII in mixtures containing kallikrein and HMW kininogen was shown by: (1) the proteolytic cleavage of factor XII; (2) the development of factor XII. coagulant activity; and (3) the proteolytic cleavage of 125l-factor XI. Platelets treated with collagen or thrombin were shown by both coagulant assays and by cleavage studies to participate with HMW kininogen and kallikrein in the proteolytic activation of factor XI by mechanisms that were partially dependent upon and partially independent of factor XII. That these zymogen activations are platelet-related was demonstrated by: (1) the enhanced coagulant activity and proteolysis observed after platelet stimulation with ADP, collagen or thrombin; (2) the inhibition of proteolytic activation seen when platelet responses were inhibited by indomethacin; (3) the absence of proteolytic activation in the absence of platelets or other surfaces; and (4) the localization of cleavage products of factor XI in platelet pellets. Experiments with celite or with unstimulated platelets present showed a linear relationship between factor-XII coagulant activity and proteolysis of factor XII. However, with activated platelets present the coagulant activity was two to fourfold greater than that observed for an equivalent amount of factor XII cleaved with celite present. These studies demonstrate that platelets can promote the proteolytic activation of factor XII by kallikrein and of factor XI by both factor-XII dependent and factor-XII-independent mechanisms.

Growth and morphology of rabbit marginal vessel endothelium in cell culture.

A procedure for the isolation and cultivation of endothelium from the marginal vessels of the rabbit ear is described. Endothelial cells, isolated by slow perfusion with a trypsin solution, are cultured in minimal essential medium supplemented with 10% fresh rabbit serum for up to 6 mo. In primary culture, marginal vessel endothelial cells grow in an expanding circular pattern with closely apposed cell membranes. Weibel-Palade bodies, subcellular organelles unique to endothelial cells in situ, are present in both primary and in serially cultivated cells (12 passages). In intact skin, Weibel-Palade (W-P) bodies are observed in the perinuclear cytoplasm in close proximity to the cell membrane facing the vascular lumen. 8-16 tubules of 200 A diameter are present in each body. In primary and subcultured cells, W-P bodies of identical size are seen in the vicinity of the Golgi apparatus and in close proximity to the outer cell membrane. At the optimum serum concentration (10%), a cell doubling time of 72-96 h is observed. When growth in normal rabbit serum and in platelet-poor serum is compared, a slower growth rate is observed in the absence of platelets, suggesting that factors released by platelets affect endothelial cell proliferation. However, addition of crude platelet factor does not substitute for complete serum. Fibroblast growth factor is not mitogenic for rabbit marginal vessel endothelium in vitro.

Vasoconstrictor response to arachidonic acid in the isolated hind limb of the dog.

1 Arachidonic acid(AA) (25-200 mug/kg) produced a dose-related increase in perfusion pressure in dog isolated hind limbs perfused with either blood or a platelet-free perfusate. 2 Prostaglandin E2 produced vasodilation while prostaglandin F2 alpha produced no vascular change at these administered doses. 3 Phentolamine did not alter the arachidonic acid response, eliminating possible alpha-adrenoceptor mediation. 4 Aspirin and idomethacin blocked the vasoconstrictor response to AA. 5 This study indicates that a vasoactive intermediate in prostaglandin synthesis can be elaborated in the absence of platelets.

Evidence for a single class of thrombin-binding sites of human platelets.

We previously demonstrated that human platelets bind a small number of thrombin molecules with high affinity and a larger number with relatively lower affinity (Tollefsen, D. M., Feagler, J. R., and Majerus, P. W. (1974), J. Biol. Chem. 249, 2646). In the present report, equilibrium binding studies using [125I]DIP-thrombin (diisopropylphosphoryl-thrombin) over the range of 0.0002-10 U/ml yield a Hill coefficient of 0.775. Measurements of the rate of dissociation of [125I]DIP-thrombin bound to platelets at relatively high affinity (0.04 U/ml added) indicate a much faster dissociation in the presence of 14 U/ml unlabeled DIP-thrombin (T 1/2 = 1.0 min) than in its absence (T 1/2 - 140 min). [125I]DIP-thrombin bound at low affinity (1.0U/ml added) dissociates from platelets with a T 1/2 = 1.7 min in the absence of unlabeled DIP-thrombin. These results suggest a negative cooperative interaction among receptor sites for thrombin; i.e., as thrombin binds to unoccupied sites, high-affinity receptors are apparently converted to low-affinity receptors. In an attempt to detect whether there is heterogeneity of thrombin receptors, [125I]DIP-thrombin was covalently cross-linked to intact platelets using 1 mM glutaraldehyde. A single complex (apparent molecular weight, 200 000) containing [125I]DIP-thrombin was formed throughout a range of thrombin concentrations in which both high- and low-affinity binding was observed. Since incorporation of [125I]DIP-thrombin into this complex did not occur in the absence of platelets and was inhibited by unlabeled thrombin, the complex may represent thrombin cross-linked to its receptor. We conclude that a single class of receptor sites can account for both high- and low-affinity binding of thrombin to platelets, although interaction between nonidentical sites cannot be excluded.

Release of nonsedimentable platelet factor 3 during coagulation.

Abstract Serum from blood clotted in the presence of platelets contains an activity originally described by O'Brien as the platelet-like activity of serum. This activity is lacking in serum obtained in the absence of platelets. It is readily detected by a modified Stypven time test and is active in enhancing prothrombin consumption in recalcified platelet-poor plasma. The activity is nonsedimentable even in high centrifugal fields (175,000 g for 2 hours) and resists denaturation at 60 °C. Part of this activity can be attributed to formation of a calcium-dependent complex between factor X and platelet factor 3 (PF-3). Factor X and PF-3 can be separated by Al(OH) 3 adsorption or by chromatography with Sephadex G-200 or G-100. Although the greater fraction of available PF-3 appears to remain with particulate platelet fragments during coagulation, between 5 and 25 per cent appears to be released in nonsedimentable form to interact with other coagulation factors.

Particle Size as a Factor in Thromboplastin Formation

Abstract Latex particles with diameters of 0.14 micron have been shown to be partially effective in the absence of platelets or phospholipids in the thromboplastin generation test.