von Willebrand factor binding to platelet GpIb initiates signals for platelet activation.

The hypothesis that von Willebrand factor (vWF) binding to platelet membrane glycoprotein Ib (GpIb) initiates intracellular pathways of platelet activation was studied. We measured the biochemical responses of intact human platelets treated with ristocetin plus vWF multimers purified from human cryoprecipitate. vWF plus ristocetin causes the breakdown of phosphatidylinositol 4,5-bisphosphate, the production of phosphatidic acid (PA), the activation of protein kinase C (PKC), increase of ionized cytoplasmic calcium ([Ca2+]i), and the synthesis of thromboxane A2. PA production, PKC activation, and the rise of [Ca2+]i stimulated by the ristocetin-induced binding of vWF multimers to platelets are inhibited by an anti-GpIb monoclonal antibody, but are unaffected by anti-GpIIb-IIIa monoclonal antibodies. Indomethacin also inhibits these responses without impairing platelet aggregation induced by vWF plus ristocetin. These results indicate that vWF binding to platelets initiates specific intraplatelet signaling pathways. The mechanism by which this occurs involves an arachidonic acid metabolite-dependent activation of phospholipase C after vWF binding to platelet membrane GpIb. This signal then causes PKC activation and increases of [Ca2+]i, which promote platelet secretion and potentiate aggregation.

Involvement of large plasma von Willebrand factor (vWF) multimers and unusually large vWF forms derived from endothelial cells in shear stress-induced platelet aggregation.

A fluid shear stress of 180 dyn/cm2 was applied for 0.5 and 5 min to platelets in citrated plasma or blood in a cone and plate viscometer with minimal platelet-surface interactions. Platelets aggregated in the shear field if large von Willebrand Factor (vWF) multimers were present. Aggregation did not require ristocetin, other exogenous agents, or desialation of vWF. Unusually large vWF multimers produced by human endothelial cells were functionally more effective than the largest plasma vWF forms in supporting shear-induced aggregation. Shear-induced aggregation was inhibited by monoclonal antibodies to platelet glycoprotein Ib or the IIb/IIIa complex, but was little affected by the absence of fibrinogen. vWF-dependent platelet aggregation under elevated shear stress in partially occluded vessels of the arterial microcirculation may contribute to thrombosis, especially if unusually large vWF multimers are released locally from stimulated or disrupted endothelial cells.

Unusually large von Willebrand factor multimers increase adhesion of sickle erythrocytes to human endothelial cells under controlled flow.

The interactions of normal erythrocytes and erythrocytes from patients having hemoglobin S hemoglobinopathies with normal human endothelial cells (EC) were investigated under flow conditions. When EC supernatant, containing 2.8-11.0 U/dl of von Willebrand factor (vWF) antigen and vWF multimeric forms larger than those present in normal plasma, was the red blood cell (RBC)-suspending medium instead of serum-free medium (SFM), the adhesion of sickle RBC, but not normal RBC, to endothelial cells was greatly increased (range of enhancement of sickle RBC adhesion, 2- to 27-fold). Adhesion of sickle RBC to endothelial cells was reduced to near serum-free levels when EC supernatant was immunologically depleted of vWF forms. Sickle RBC suspended in SFM containing 200 U/dl of purified vWF multimers of the type found in normal human plasma or 300 micrograms/ml human fibronectin were only slightly more adhesive to endothelial cells than sickle RBC suspended in SFM alone. These data indicate that unusually large vWF multimers produced by endothelial cells are potent mediators of the adhesion of sickle erythrocytes to endothelial cells. Vaso-occlusive crises in sickle cell anemia may be caused, at least in part, by adhesive interactions between the abnormal surfaces of sickle RBC and the endothelium after the release of unusually large vWF multimeric forms from stimulated or damaged endothelial cells.

Novel ADAMTS-13 mutations in an adult with delayed onset thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is associated with congenital and acquired deficiency of ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and reduces its adhesive activity. Mutations throughout the ADAMTS13 gene have been identified in congenital TTP patients, most of whom have initial episodes during infancy or in early childhood. PATIENTS AND METHODS: We report the case of an adult male who was diagnosed with idiopathic thrombocytopenic purpura at age 34, and with TTP 14 years later. The patient was compound heterozygous for an 18 bp in-frame deletion (C365del) in the disintegrin domain and a point mutation of R1060W in the seventh thrombospondin domain of the ADAMTS-13 gene. CONCLUSIONS: In vitro studies found that C365del and R1060W severely impair ADAMTS-13 synthesis in transfected Hela cells, whereas the deletion mutant also failed to cleave VWF under static and flow conditions.

Platelets adhered to endothelial cell-bound ultra-large von Willebrand factor strings support leukocyte tethering and rolling under high shear stress.

Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm(2) in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.

Platelet-derived VWF-cleaving metalloprotease ADAMTS-13.

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.

Morphine-induced immune thrombocytopenia.

Sudden thrombocytopenia that was temporally related to the administration of morphine sulfate developed in a 23-year-old woman. A morphine-dependent platelet antibody was found in her serum by chromic chloride Cr 51 platelet lysis. The antibody was complement dependent, present in the IgG immunoglobulin fraction, and its drug-dependent platelet lytic activity was demonstrable with several narcotic analgesics in addition to morphine. The antibody activity declined over an eight-month period following recovery from thrombocytopenia. To our knowledge, morphine-induced immune thrombocytopenia has not previously been described.

Progressive multifocal leukoencephalopathy: failure of response to transfer factor and cytarabine.

A 57-year-old woman with a 2-year history of neurologic symptoms was shown on brain biopsy to have progressive multifocal leukoencephalopathy. Although cell-mediated immune mechanisms were impaired, no underlying disease was found. The administration of transfer factor enhanced cell-mediated immunity, but the neurologic symptoms did not improve. Separate courses of glucocorticoids and cytarabine were associated with rapid neurologic deterioration.

A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagen.

Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb-IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb-IIIa and P-selectin than those on VWF. Activation of GpIIb-IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb-IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates.

Hemolytic anemia associated with multiple autoantibodies and low serum complement.

A 37 year old woman with extravascular hemolytic anemia had a positive Monospot test associated with positive antiglobulin and anticomplement Coombs' tests, cold agglutinins and warm autoantibodies. IgG-kappa (k) antibodies, which reacted with all panel red cells at 37 degrees C, were eluted from her circulating red cells. However, neither immunoglobulins nor C3 was detected after her serum was adsorbed with heterologous red cell stroma at 37 degrees C and eluted at the same temperature in glycine buffer. In contrast, IgM-kappa and IgM-lambda (lambda), IgG-3-kappa, IgG4-lambda, IgA-lambda and C3 were eluted at 37 degrees C from heterologous red cell stroma after adsorption with her serum at 0 degrees C. Thus, antibodies of several types, which were present in the patient's serum, reacted optimally with red cell antigens at low temperature. Cold-reactive IgG3-kappa antibodies, which also capable of interacting with red cells at 37 degrees C, probably accounted for the IgG-kappa antibodies eluted from the patient's circulating red cells. The patient's serum C4 titers were decreased, with low normal to moderately depressed C3 and low normal C5, indicating that the anti-red cell IgM and/or IgG3-kappa antibodies probably fixed complement. A localized cold stress test resulted in a transient increase in plasma hemoglobin and a decrease in serum C3 titer. These findings, and the beneficial clinical response obtained with small doses of prednisone, suggest that both the cold-reactive antibodies and the IgG-kappa on circulating red cells were pathophysiologically significant. This is the first report of a patient with multiple red cell autoantibodies in whom serum complement component titers were determined in conjunction with characterization of the anti-red cell immunoglobulins. Subclinical infectious mononucleosis may have preceded the prolonged hemolytic episode. Clinical evidence of systemic lupus erythematosus has not appeared.

Sickle cell--betao thalassemia variant with high hemoglobin F and mild clinical course.

A 70 year old Black woman had chronic hemolytic anemia without recurrent painful crises. Hemoglobin pattern by electrophoresis was hemoglobin S (69 to 71 per cent), hemoglobin A2 (4.6 per cent) and hemoglobin F (24 to 27 per cent). No hemoglobin A was detected, and the hemoglobin F was distributed heterogeneously in the red cells. Reticulocyte alpha/nonalpha globin chain synthetic ratios were 1.44 to 1.62. Thus, the patient had a high hemoglobin F variant of S-beta zero (betao) thalassemia which has not been described previously. Her clinical course has been mild in comparison with S-betao thalassemia patients who do not have extremely elevated hemoglobin F levels.

Abnormalities of von Willebrand factor multimers in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

PURPOSE: To analyze and review von Willebrand factor (vWF) multimeric patterns in patients with single-episode thrombotic thrombocytopenic purpura (TTP), intermittent TTP (episodes at infrequent, irregular intervals), chronic relapsing TTP (episodes at frequent, regular intervals), and the hemolytic-uremic syndrome (HUS). PATIENTS AND METHODS: Platelet-poor plasma samples were obtained in EDTA, citrate, or citrate-hirudin-aprotinin-leupeptin from 36 patients with single-episode TTP, eight patients with intermittent TTP, four patients with chronic relapsing TTP, and 26 patients with HUS. The samples were separated by sodium dodecyl sulfate-agarose gel electrophoresis, overlaid with rabbit 125I-anti-human vWF IgG, and analyzed by autoradiography. RESULTS: Abnormalities of vWF multimers were found in platelet-poor plasma samples from 31 of 36 found in platelet-poor plasma samples from 31 of 36 patients (86%) at the onset of and during their single TTP episode. vWF multimers larger than those in normal plasma, and similar to vWF forms observed within normal human endothelial cells (unusually large vWF multimers), were demonstrated in 31% of the patients; 19% had either unusually large vWF multimers or a relative decrease in the largest plasma vWF forms in different serial samples; 36% had a relative decrease in the largest plasma vWF forms. These results imply that endothelial cell injury or intense stimulation, along with the attachment of unusually large vWF multimers and the largest plasma vWF forms to platelets, occurred during the single TTP episodes in most patients. Patterns of vWF multimers were normal in 92% of patients with single-episode TTP studied after recovery. All eight patients with intermittent TTP and the four patients with chronic relapsing TTP had unusually large vWF multimers in their plasma between episodes, and these multimers decreased or disappeared during relapses. Of 26 children and adults with HUS, 14 had a relative decrease in the largest plasma vWF multimeric forms and one had unusually large vWF multimers during the episode (vWF multimeric abnormalities in 58% of the patients). CONCLUSION: It is probable that vWF was involved in the pathophysiology of TTP in most of these patients with the single-episode, intermittent, or chronic relapsing types of TTP, and in more than 50% of the patients with HUS.

Alterations in hemostatic parameters during hemodialysis with dialyzers of different membrane composition and flow design. Platelet activation and factor VIII-related von Willebrand factor during hemodialysis.

The effect of dialyzer membrane and design on hemostatic parameters during hemodialysis were evaluated in a prospective controlled study. This study demonstrated that hemodialysis is associated with significant platelet activation and loss, which are influenced by both dialyzer configuration and membrane composition. In addition, use of the cuprophan membrane is associated with greater perturbations of the vascular endothelium, as reflected in changes in factor VIII-related von Willebrand factor and 6-keto-prostaglandin F1 alpha concentrations not seen with the polyacrylonitrile membrane. Of the dialyzers studied, the polyacrylonitrile membrane in a hollow-fiber configuration appears to minimize platelet loss and activation, and to minimize increases in factor VIII-related von Willebrand factor and 6-keto-prostaglandin F1 alpha.

Thrombotic thrombocytopenic purpura.

Recent studies indicate that CRTTP patients have excessive shear stress-induced platelet aggregation that is associated with the presence of ULvWF multimers in their plasma and increased vWF-binding to their platelets by flow cytometry. In these CRTTP patients, relapses, excessive shear-aggregation and the presence in their plasma of ULvWF forms are all reversed by the infusion of normal FFP or substances devoid of the largest vWF multimers found in plasma (cryoprecipitate-poor plasma or cryosupernatant; solvent/detergent-treated plasma) without the need for concurrent plasmapheresis. This constellation of observations, along with reports of increased vWF in TTP platelet thrombi, increases the probability that ULvWF multimers derived from injured or abnormal endothelial cells induce aggregation during TTP episodes in high-shear regions of the microcirculation.

Differential effects of cytochalasin B on platelet release, aggregation and contractility: evidence against a contractile mechanism for the release of platelet granular contents.

Cytochalasin B alters the structure and functional properties of filamentous actin. Platelet-mediated clot retraction in dilute platelet-rich plasma (PRP) is inhibited progressively at cytochalasin B concentrations of 0.01 mg/ml, 0.05 mg/ml and 0.1 mg/ml. Dynamic rheological measurements of recalcified PRP in a Weissenberg Rheogoniometer indicate that platelet contractility (as reflected in measurements of elastic moduli) is reduced by 33%, 57% and 63% at cytochalasin B concentrations of 0.01, 0.05 and 0.1 mg/ml, respectively. In contrast, pre-incubation of human platelet-rich plasma (PRP) with 0.01 mg/ml or 0.05 mg/ml cytochalasin B does not inhibit collagen-induced [14C]serotonin release on collagen-induced-platelet aggregation, which is dependent on the release of ADP from platelet dense granules. Even at a cytochalasin B concentration of 0.1 mg/ml, collagen-induced [14C-]serotonin release and aggregation are impaired only moderately. Cytochalasin B does not interfere with the uptake by platelets of [14C-]-serotonin, or with the kinetics and extent of clot formation in platelet-free plasma. Thus, concentrations of cytochalasin B which impair platelet contractility do not inhibit the release of platelet dense granule contents. It is concluded that neither the platelet release reaction nor platelet aggregation is dependent on platelet contractile mechanisms.

Effects of prostaglandins, derivatives of cyclic 3':5'-AMP, theophylline, cholinergic agents and colchicine on clot retraction in dilute platelet-rich plasma and gel-separated platelet test systems.

In dilute suspensions of platelet-rich plasma (PRP) or gel-separated platelets (GSP), dibutyryl-cAMP (DBcAMP) and monobutyryl-cAMP inhibited platelet-mediated fibrin clot retraction in concentrations of 2--3 X 10(-6) M, with complete inhibition at 1--3 X 10(-4) M. Prostaglandin E1 (PGE1), which inhibited fibrin clot retraction in concentrations greater than 1.5--3 X 10(-8) M, was a more effective inhibitor than either PGE2 or PGF2 alpha. In the presence of theophylline (10-4 M), concentrations of DBcAMP, PGE1, PGE2 and PGF2 alpha necessary to inhibit fibrin clot retraction were reduced 50-fold for DBcAMP and 2.5 to 20-fold for the prostaglandins. In dilute PRP or GSP, inhibition of fibrin clot retraction does not result from inhibition of thrombin-induced platelet aggregation. Thus, compounds which increase platelet cAMP levels result in the inhibition of platelet-mediated fibrin clot retraction, and this inhibitory effect may be mediated, at least in part, through suppression of platelet contractility. Cyclic GMP, dibutyryl-cGMP and carbamylcholine-Cl (which stimulate guanylate cyclase) did not influence fibrin clot retraction, and did not prevent inhibition of fibrin clot retraction by DBcAMP and PGE1. Colchicine, in concentrations known to disrupt platelet microtubules (2.5 X 10(-6) M to 2.5 X 10(-3) M), had little inhibitory effect on either fibrin clot retraction or platelet (3H)-serotonin release.

Fibrillin containing elastic microfibrils support platelet adhesion under dynamic shear conditions.

The vascular subendothelium contains macromolecular structures called microfibrils. Type VI collagen is one protein found in microfibrils that supports platelet adhesion and aggregation and we have previously evaluated the roles of platelet receptors and vWf involved in these processes under physiological shear conditions. Here we investigate the ability of fibrillin containing elastic microfibrils to support mural thrombus formation. Our results show that elastic microfibril surfaces support platelet adhesion under low shear conditions at a level similar to collagen VI tetramers. However, the degree of aggregation on the elastic microfibril surface is much higher. Both adhesion and aggregation were shown to be mediated by the GPIIb-IIIa platelet receptor. Elastic microfibrils do not support the formation of mural thrombi under high shear conditions. These results suggest roles for both collagen VI and fibrillin containing elastic microfibrils in modulating the platelet response to blood vessel injury.

Intrathoracic extramedullary hematopoiesis: report of a case in a patient with sickle-cell disease-beta-thalassemia.

A case of paravertebral extramedullary hematopoiesis in a patient with sickle-cell anemia-beta-thalassemia is reported. Interesting aspects in this case, including the high level of Hb F and large number of nucleated erythrocytes in the peripheral blood, are discussed. Fifty-five cases of intrathoracic extramedullary hematopoiesis have been reported. Most of these cases occurred in patients with thalassemia or hereditary spherocytosis. Extramedullary hematopoiesis should be considered in the differential diagnosis of a posterior mediastinal mass in a patient who has chronic anemia or other syndromes associated with extramedullary hematopoiesis.

Postpartum thrombotic thrombocytopenic purpura complicated by Budd-Chiari syndrome.

BACKGROUND: Thrombotic thrombocytopenic purpura is an infrequent but devastating complication of pregnancy, often difficult to differentiate from severe preeclampsia and the syndrome of hemolysis, elevated liver enzymes, and low platelets (HELLP). To our knowledge, the combination of thrombotic thrombocytopenic purpura and hepatic vein thrombosis has not been reported previously in pregnancy. CASE: A 33-year-old woman, a multigravida, was delivered at 36 weeks' gestation because of pregnancy-induced hypertension and HELLP syndrome. Postpartum, the patient became obtunded, disoriented, and anuric. Her laboratory values revealed a Coombs-negative, microangiopathic hemolytic anemia, modestly abnormal coagulation studies, and thrombocytopenia. She also had elevated liver enzymes, lactate dehydrogenase, and creatinine. The largest plasma von Willebrand factor multimeric forms were relatively decreased in her ethylenediaminetetra-acetic acid-platelet-poor plasma. A diagnosis of thrombotic thrombocytopenic purpura was made. The patient received plasma exchange, hemodialysis, blood transfusion, and glucocorticoids. She responded to therapy, but was later noted to have increasing hepatosplenomegaly, rising levels of bilirubin, and elevated alkaline phosphatase. A Doppler study and magnetic resonance imaging demonstrated the absence of flow in the middle and left hepatic veins, secondary to thrombosis (Budd-Chiari syndrome). She was maintained on warfarin therapy and was discharged on postpartum day 50. CONCLUSION: The early recognition of thrombotic thrombocytopenic purpura is crucial for the initiation of appropriate treatment as soon as possible. The avoidance of platelet transfusion and early plasma-exchange therapy may be lifesaving.

von Willebrand factor multimeric levels and patterns in patients with severe preeclampsia.

The clinical manifestations of severe preeclampsia and thrombotic thrombocytopenic purpura are similar. Patients with thrombotic thrombocytopenic purpura have been demonstrated to have larger than usual von Willebrand factor multimers. Serial serum samples were taken from patients with severe preeclampsia, all with platelet counts less than 50,000. Von Willebrand factor multimeric patterns were normal in all the patients except one, who later proved to have chronic relapsing thrombotic thrombocytopenic purpura. All patients had elevated levels of von Willebrand factor. Although the clinical presentations of thrombotic thrombocytopenic purpura and severe preeclampsia with thrombocytopenia have many similarities, the underlying pathophysiology of each disorder appears to be different.