The Haemostatic Effect of 51Cr-Labelled Blood Platelets An Experimental Study in the Rabbit

The haemostatic effect of ⁵¹Cr-labelled platelets was studied in 5 rabbits made thrombocytopenic (35,000/μl blood) by whole body ionizing irradiation. Bleeding times were recorded after standardized cuts on the inner side of the rabbit's ear, a method with an acceptable reproducibility. The animals were then each transfused with concentrates of labelled platelets from 2 healthy donor rabbits. This increased the platelet counts to about 2 times 10⁵/μl blood. Bleeding time values were markably prolonged before transfusion and became normalized when tested 1 and 4 h after transfusion. In 3 control experiments, where unlabelled platelet rich plasma was transfused to thrombocytopenic recipients, a similar shortening of the bleeding time was observed. It is concluded that ⁵¹Cr-labelled platelets retain haemostatic ability comparable to non-labelled platelets, when circulating in a recipient animal.     

Pathogenesis of thrombocytopenia in cyanotic congenital heart disease

Although a significant minority of patients with cyanotic congenital heart disease (CCHD) are thrombocytopenic, the pathogenesis and prevalence have not been established. This study was designed to address these 2 issues. We included 105 patients with CCHD (60 men and 45 women; aged 21 to 54 years). Systemic arterial oxygen saturations were 69% to 78%. Hematocrits were 62% to 74% with normal iron indexes. In 26 of 105 patients (25%), platelet counts were <100x10(9)/L. The diagnosis was Eisenmenger syndrome in all 26 patients with thrombocytopenia. Platelet production was determined by flow cytometric reticulated platelet counts. Megakaryocyte mass was determined indirectly by thrombopoietin levels. Disseminated intravascular coagulation was based on prothrombin time, activated partial thromboplastin time, and D-dimers. Platelet activation was determined by levels of platelet factor 4 and beta thromboglobulin. Reference ranges were derived from 20 normal acyanotic controls. A reduction in absolute reticulated platelet counts implied decreased platelet production (p<0.001). Normal thrombopoietin levels implied normal megakaryocyte mass. Normal prothrombin time, activated partial thromboplastin time, and D-dimers excluded disseminated intravascular coagulation. Normal platelet factor 4 and beta thromboglobulin indicated absent or minimal platelet activation. Twenty-five percent of the patients with CCHD were thrombocytopenic because platelet production was decreased despite normal megakaryocyte mass. We hypothesized that right-to-left shunts deliver whole megakaryocytes into the system arterial circulation, bypassing the lungs where megakaryocytic cytoplasm is fragmented into platelets, thus reducing platelet production. In conclusion, platelet counts in CCHD appear to represent a continuum beginning with low normal counts and ending with thrombocytopenia.     

Ultrastructural changes of endothelium associated with thrombocytopenia.

In a study of the relationship between thrombocytopenia and increased vascular fragility, changes in the endothelium of capillaries and postcapillary venules of the tongue were examined by electron microscopy. Adult male albino rabbits (4 kg) were maintained thrombocytopenic (platelets less than 20,000/cu mm) up to 24 hr by one to three injections of guinea pig antirabbit platelet serum. Within 6 hr the normal projections and folds of the lumenal surface of the endothelial surface were largely effaced. In addition, the endothelium became thinner. In places, pores and membranous diaphragms were observed. Endothelial junctions appeared normal. Identical findings were observed if rabbits were made thrombocytopenic by administration of intraperitoneal busulfan. Intravenously administered Thorotrast was observed in endothelial cells and in the extravascular spaces within 3 min after injection into thrombocytopenic animals, while it was seen only intravascularly in control rabbits. With the spontaneous restoration of circulating platelets, the endothelium reverted to normal.     

Effects of recombinant human megakaryocyte growth and development factor on platelet activation

The c-Mpl receptor for thrombopoietin and its recombinant related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF on platelet aggregation in platelet-rich plasma (PRP) and in whole blood. The results obtained indicate that MGDF by itself did not affect platelet aggregation. However, when added before other agonists such as adenosine diphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of MGDF was dependent on the dose and on the time of platelet preincubation, and it occurred both in PRP and in whole-blood platelet aggregation. MGDF also "primed" the release of adenosine triphosphates and the production of thromboxane B2 by platelets stimulated with ADP, EPI, and THR. When added 15 seconds after the preincubation of platelets with subthreshold concentrations of ADP, EPI, and THR, MGDF exhibited a synergism with these agonists. Moreover, we observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential.     

Thrombopoietin contributes to enhanced platelet activation in patients with unstable angina.

OBJECTIVES: We sought to investigate the potential role of elevated levels of thrombopoietin (TPO) in platelet activation during unstable angina (UA). BACKGROUND: Thrombopoietin is a humoral growth factor that does not induce platelet aggregation per se, but primes platelet activation in response to several agonists. No data concerning its contribution to platelet function abnormalities described in patients with UA are available. METHODS: We studied 15 patients with UA and, as controls, 15 patients with stable angina (SA) and 15 healthy subjects. We measured TPO and C-reactive protein (CRP), as well as monocyte-platelet binding and the platelet expression of P-selectin and of the TPO receptor, c-Mpl. The priming activity of patient or control plasma on platelet aggregation and monocyte-platelet binding and the role of TPO in this effect also were studied. RESULTS: Patients with UA showed higher circulating TPO levels, as well as increased monocyte-platelet binding, platelet P-selectin expression, and CRP levels, than those with SA and healthy control subjects. The UA patients also showed reduced platelet expression of the TPO receptor, c-Mpl. In vitro, the plasma from UA patients, but not from SA patients or healthy controls, primed platelet aggregation and monocyte-platelet binding, which were both reduced when an inhibitor of TPO was used. CONCLUSIONS: Thrombopoietin may enhance platelet activation in the early phases of UA, potentially participating in the pathogenesis of acute coronary syndromes.     

Endogenous thrombopoietin levels during the clinical management of acute myeloid leukaemia

Thrombocytopenia represents a major problem in the management of acute myeloid leukaemia (AML). The data regarding the alterations of endogenous thrombopoietin (TPO) regulation during the clinical course of AML are limited. The aim of this study was to investigate endogenous TPO dynamics in association with platelets during the clinical course of AML. We serially measured both TPO and platelets concurrently over the entire treatment period of newly diagnosed patients receiving both remission induction and consolidation chemotherapies. The median concentration of TPO in AML patients at the initial diagnosis was 469.71 pg/ml and increased significantly during the aplastic period due to remission induction chemotherapy (median: 1085.33 pg/ml) but then decreased to a level (median: 45.26 pg/ml) encountered in the healthy control subjects (median: 56.90 pg/ml). In the cytopenic period due to consolidation treatment, TPO level again increased significantly to a high level (median: 891.38 pg/ml) during the platelet nadir, but decreased toward normal (median: 100.75 pg/ml) after the thrombocytopenic period had elapsed. In conclusion, endogenous TPO levels exhibit an inverse fluctuation in relation to platelet counts during the clinical course of AML. Pharmacological stimulation of thrombopoiesis in AML with novel molecules, including the recombinant thrombopoietins and the small peptide agonists, should be based on a critical administration strategy that must consider the endogenous levels of TPO. TPO levels in distinct AML disease states may explain the unsuccessful recombinant TPO trials and could help to design better strategies for 'pharmacological stimulation of thrombopoiesis' in AML.     

Comparison of glycocalicin,thrombopoietin and reticulated platelet measurement as markers of platelet turnover in HIV+ samples

It is important to distinguish between decreased platelet/megakaryocyte production or increased peripheral platelet destruction as causes of thrombocytopenia. The measurement of reticulated platelets, plasma glycocalicin and thrombopoietin (TPO) levels are potentially of use as discriminators. Thrombocytopenia occurs in many HIV+ patients, and plasma glycocalicin has previously been shown to be elevated in this patient group. Reticulated platelets, glycocalicin and TPO were measured in samples from 56 HIV+ subjects and 20 healthy normal controls. The glycocalicin index (GCI - the glycocalicin levels adjusted for the platelet count) measured in HIV+ subjects was found to be significantly elevated when compared to normal controls (mean GCI 1.5 and 1.27, p = 0.04), while the percentage of reticulated platelets and TPO levels were not. Thrombocytopenic HIV+ subjects had significantly elevated mean GCI (2.8 and 1.4, p < 0.0001), TPO (85.2 and 27.2 pg/ml, p = 0.002), percentage of reticulated platelets (15.3 and 10.8%, p = 0.01), and significantly reduced absolute numbers of reticulated platelets (16.2 and 24.5 2 10 9 /l, p = 0.0004) when compared to non-thrombocytopenic HIV+ subjects. GCI and percentage of reticulated platelets exhibited a significant positive correlation ( r = 0.4, p = 0.002) in HIV+ subjects. The reticulated platelet, TPO and GCI data suggests that thrombocytopenic HIV+ subjects have normal platelet production, and increased peripheral platelet destruction.     

Comparison of glycocalicin, thrombopoietin and reticulated platelet measurement as markers of platelet turnover in HIV+ samples

It is important to distinguish between decreased platelet/megakaryocyte production or increased peripheral platelet destruction as causes of thrombocytopenia. The measurement of reticulated platelets, plasma glycocalicin and thrombopoietin (TPO) levels are potentially of use as discriminators. Thrombocytopenia occurs in many HIV+ patients, and plasma glycocalicin has previously been shown to be elevated in this patient group. Reticulated platelets, glycocalicin and TPO were measured in samples from 56 HIV+ subjects and 20 healthy normal controls. The glycocalicin index (GCI--the glycocalicin levels adjusted for the platelet count) measured in HIV+ subjects was found to be significantly elevated when compared to normal controls (mean GCI 1.5 and 1.27, p = 0.04), while the percentage of reticulated platelets and TPO levels were not. Thrombocytopenic HIV+ subjects had significantly elevated mean GCI (2.8 and 1.4, p < 0.0001), TPO (85.2 and 27.2 pg/ml, p = 0.002), percentage of reticulated platelets (15.3 and 10.8%, p = 0.01), and significantly reduced absolute numbers of reticulated platelets (16.2 and 24.5 x 10(9)/l, p = 0.0004) when compared to non-thrombocytopenic HIV+ subjects. GCI and percentage of reticulated platelets exhibited a significant positive correlation (r = 0.4, p = 0.002) in HIV+ subjects. The reticulated platelet, TPO and GCI data suggests that thrombocytopenic HIV+ subjects have normal platelet production, and increased peripheral platelet destruction.     

Cardiopulmonary dysfunctions caused by intravenous collagen-induced release of ADP and ATP from platelets.

Bovine tendon collagen suspension (4.5 mg/kg) was injected rapidly into the femoral vein of 14 normal (untreated) and 8 busulfan treated rats. Trasient effects included decreased platelet counts and arterial PO2, increased central venous pressure, apnea, bradycardia and variable A-V block. These findings were most prominent within 1 minute after injection and subsided or disappeared by 10 minutes. During this period, ADP and ATP in platelet-free plasma from carotid arterial blood were measured in a liquid scientillation counter using the firefly luciferase assay. In normal (untreated) rats, collagen injection was followed by increases in plasma ADP and ATP, a rise in plasma hemoglobin and minimal changes of fibrinogen and hematcrit. Pathological observations indicated the platelet emboli in pulmonary vessels. In contrast, rats made thrombocytopenic by intraperitoneally infected busulfan prior to collagen injection had minimal or no change in platelet count, plasma ADP, ATP, hemoglobin, fibrinogen, or cardiopulmonary functions following collagen injection. These findings suggest that collagen injection causes release of ADP and ATP from platelets; released ADP induces platelets to form aggregates which lodge in the coronary and pulmonary microcirculations and elsewhere, resulting in thrombocytopenia and the cardiopulmonary dysfunction, in the presence of shear, of red cells with vessel surfaces altered by platelet aggregates.     

Pathophysiology and thrombokinetics in autoimmune thrombocytopenia

Studies that have measured platelet survival by autologous platelet labeling with (111)In and (51)Cr have differed in their results. Although all studies have revealed a significant decrease in platelet life span, the rates of platelets entering the circulation, a calculated and inferred determination, have been found to be moderately decreased to as much as five times normal. Several mechanisms have been proposed to explain an apparent decrease in platelet production, including a true decrease due to damage to the megakaryocytes by autoantibody, versus a decrease only in 'effective' production due to intramedullary destruction by the reticuloendothelial system. Recently, the identification of the cytokine, thrombopoietin, has allowed the evaluation of another aspect of the pathophysiology of thrombocytopenic states. Megakaryocyte growth factor levels are increased 10 to 20 times in patients who are thrombocytopenic due to chemotherapy or aplastic anemia, but may be decreased, normal, or only modestly raised in patients with immune platelet destruction. Autologous platelet survival measurements, prior to and while on therapy with a stable platelet count, reveal that removal of part of the reticuloendothelial system with splenectomy leads to increased platelet survival and platelet number. Similar studies reveal that corticosteroid treatment for immune thrombocytopenic purpura (ITP) effectively increases the rate of platelet production but does not change platelet survival. It may be that other therapies are effective by a combination of these mechanisms. Stimulation of thrombopoietin production or administering exogenous cytokine may have a role to play in future management of patients with ITP.     

Thrombocytopenia in HIV infection: impairment of platelet formation and loss correlates with increased c-Mpl and ligand thrombopoietin expression

Thrombocytopenia is a common hematologic disorder in patients infected with the human immunodeficiency virus (HIV) and represents a risk for bleeding which is further deleterious during surgery. The major causes of the thrombocytopenia include accelerated peripheral platelet destruction by antiplatelet antibodies and insufficient production of platelets from the infected megakaryocytes. Additionally, at an earlier stage of platelet development, HIV may inhibit megakaryopoiesis at multiple stages of pluripotent CD34+ progenitor stem cell differentiation possibly contributing to decreased levels of platelets in circulation. In HIV infected patients, both the serum thrombopoietin (TPO) levels and theTPO-c-Mpl complexes on the platelet surface were significantly elevated. Therapeutic infusion of HIV infected patients with pegylated recombinant human megakaryocyte growth development factor (PEG-rHu-MGDF) restores platelet counts to normal levels and reduces the c-Mpl expression per platelet. In vitro aggregation of platelets treated with TPO and agonist, adenosine diphosphate (ADP), decrease the dose of ADP that is required for half-maximum aggregation. In vivo dosing does not effect platelet aggregation showing that the metabolism of TPO following its internalization through TPO-c-Mpl complex is rapid and that dosing within the therapeutic range does not constitute increased risk of thrombotic disease.     

Type IIB von Willebrand's disease associated with a complex thrombocytopenic thrombocytopathy

A familial bleeding disorder characterized by an association of Type IIB von Willebrand's disease (vWD) with a complex thrombocytopenic thrombocytopathy is described in two patients from the same generation. Findings typical of type IIB vWD included enhanced ristocetin-induced binding of patient von Willebrand factor (vWF) to platelets of patients and normal individuals in association with the absence of larger multimers from plasma. Abnormalities in platelet function included deficient platelet aggregation to ADP, collagen, epinephrine, and arachidonic acid; and defective release of 14C-serotonin, vWF, and platelet factor 4 (PF4) in response to thrombin, collagen, or ADP. Platelet factor 4 and platelet vWF were decreased when measured per mg of total platelet protein. In addition, the binding of normal vWF to patient platelets stimulated with thrombin was decreased. Platelet size was increased with a very heterogeneous distribution width. Electron microscopic evaluation showed giant platelets with dense and alpha bodies present. The platelet count was borderline or slightly decreased in the resting state and declined to frankly thrombocytopenic levels at the time of acute bleeding episodes; this state was associated with the presence of platelet aggregates in blood smears.     

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.     

Thrombopoietic cytokines in relation to platelet recovery after bone marrow transplantation

In order to evaluate the importance of different thrombopoietic stimulatory cytokines in accelerating platelet recovery after bone marrow transplantation (BMT), we assayed serial plasma concentrations of three cytokines, thrombopoietin (TPO), interleukin-6 (IL-6), and IL-11 through the course of platelet nadir and recovery after BMT. Both mean TPO and IL-6 levels showed a marked rise and later fall preceding or coincident with the platelet nadir and recovery, suggesting their potential role as circulating regulators or stimulators of thrombopoiesis. In contrast, IL-11 levels remained remarkably constant through the whole course suggesting that this cytokine, though capable of stimulating thrombopoiesis, does not serve as a circulating regulator of platelet production. Additionally, we assayed the levels of these three cytokines following initial platelet transfusion to assess the capacity of transfused platelets to adsorb these thrombopoietic cytokines from the plasma and reduce their circulating levels, thus potentially modifying their availability for stimulating megakaryocyte proliferation. No consistent falls in TPO, IL-6 or IL-11 levels were observed following the initial two platelet transfusions. These data support the importance of circulating TPO and IL-6 as hormones capable of stimulating platelet production. Their physiologic relevance as in vivo regulators of thrombopoiesis and clinical utility for therapy of thrombocytopenia need further investigation.     

Relationships between thrombopoiesis and erythropoiesis: with studies of the effects of preparations of thrombopoietin and erythropoietin

The effects of administration of partially purified human urinary erythropoietin and rabbit thrombopoietin, and of endogenously produced erythropoietin and thrombopoietin on both red cell and platelet production were examined in mice. Partially purified thrombopoietin was prepared from rabbit plasma by sequential fractionation with ammonium sulfate precipitation, and DEAE and Sephadex G-100 chromatography. Preparations of thrombopoietin and partially purified human urinary erythropoietin (NIH No. H-11-TaLSL) were administered subcutaneously to normal mice, and the rate of incorporation of selenomethionine-75 Se into platelets was measured as an index of thrombopoietic activity of the infused material. Erythropoietin and thrombopoietin were assayed for erythropoietic activity by measuring the rate of appearance of 59Fe in the red cells of posthypoxic polycythemic mice. Preparations containing thrombopoietin had barely measurable erythropoietic activity, and 7 units of partially purified erythropoietin had little thrombopoietic activity. When endogenous levels of erythropoietin were increased by hypoxia, platelet production was not enhanced. Similarly, increased levels of thrombopoietin, induced in response to thrombocytopenia produced by platelet antiserum, did not alter red cell production. These data suggest that physiologically increased levels of thrombopoietin do not stimulate erythropoiesis, and that physiologically increased levels of erythropoietn do not stimulate thrombopoiesis. However, currently available, partially purified preparations of erythropoietin and thrombopoietin may be capable of stimulating both platelet and red cell production if used in sufficient quantities.     

A Comparison of Platelet Production in Mice made Thrombocytopenic by Hypoxia and by Platelet Specific Antisera

S ummary . Previous experiments have shown that hypoxia causes thrombocytopenia in mice. In an attempt to define the recovery phase, mice were enclosed in hypoxia chambers for 14 d and platelet counts, total circulating platelet counts (TCPC) and masses (TCPM), platelet sizes, and %³⁵S incorporation into platelets were determined over the next 8 d while the mice were kept at ambient O₂ levels. For comparison, untreated mice and mice made thrombocytopenic by injection of rabbit anti-mouse-platelet serum (RAMPS) were used. Both 14 d of hypoxia and RAMPS treatment resulted in severe thrombocytopenia. TCPC and TCPM were similar in both exhypoxic and RAMPS-injected mice. However, the platelet count recovery pattern of exhypoxic-thrombocytopenic mice did not show the rebound-thrombocytosis which was characteristic of RAMPS-induced thrombocytopenia, apparently because of dilution of platelets by increased blood volumes. Average platelet sizes were larger than normal 2 d posthypoxia or after RAMPS-treatment, followed by a decline toward normal; platelets from exhypoxic mice, however, remained larger. Per cent ³⁵S incorporation into platelets was lower in exhypoxic mice than in RAMPS-treated mice; lower numbers of megakaryocytes were observed immediately after removal from hypoxia followed by an increase in number and size by 2 d later. Also, thrombopoietin was detected in plasma of RAMPS-treated mice, but not in plasma of exhypoxic-thrombocytopenic mice. Therefore, it seems possible that hypoxia reduced the numbers of megakaryocytes, resulting in depressed thrombocytopoiesis of mice at the time of removal from hypoxic atmospheres.     

p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin

To investigate the signaling processes induced by recombinant thrombopoietin, we used human platelets to recently show that thrombopoietin induces rapid tyrosine phosphorylation of Jak2, Tyk2, Shc, Stat3, Stat5, and other proteins in human platelets. Because the apparent molecular weight of a major tyrosine-phosphorylated protein in platelets stimulated by thrombopoietin is approximately 120 kD, we examined the possibility that this could be p120c-cbl, a protein known to be involved in signaling by many growth factors. Specific antisera against p120c-cbl recognized the same 120-kD protein in lysates of Jurkat cells, which are known to express p120c-cbl, and platelets, indicating that platelets have p120c-cbl. Thrombopoietin induced rapid tyrosine phosphorylation of p120c-cbl in platelets. Thrombopoietin also induced tyrosine phosphorylation of p120c-cbl in FDCP cells genetically engineered to express the thrombopoietin receptor, c-Mpl. Interestingly, FDCP cells, expressing a truncated c-Mpl devoid of the box-2 domain, proliferate in response to thrombopoietin. However, no increase in tyrosine phosphorylation of p120c-cbl was observed upon treatment of these cells with thrombopoietin, indicating that in this system tyrosine phosphorylation of p120c-cbl may not be essential for cell proliferation. This suggests that tyrosine phosphorylation of p120c-cbl may be required for nonmitogenic responses induced by thrombopoietin in postmitotic cells such as platelets. On the other hand, p120c-cbl was not significantly tyrosine-phosphorylated upon treatment of platelets with thrombin. However, it became incorporated into the Triton X-100-insoluble, 10,000g-sedimentable residue in an aggregation-dependent manner, suggesting that it may have a regulatory role in platelet cytoskeletal processes. p120c-cbl was constitutively associated with a 28-kD adapter protein, Grb2, that was also incorporated into the Triton X-100-insoluble, sedimentable residue dependent on aggregation. Further, we found that p120c-cbl is an endogenous substrate for calpain, a protease that may play a role in postaggregation signaling processes. Our data suggest that p120c-cbl may be involved in signal transduction following ligand binding to c- Mpl through its inducible tyrosine phosphorylation, and it may also be involved in signaling during platelet aggregation by its redistribution to the cytoskeleton.