Eberhard Morgenstern

Intravascular tissue factor initiates coagulation via circulating microvesicles and platelets

Although tissue factor (TF), the principial initiator of physiological coagulation and pathological thrombosis, has recently been proposed to be present in human blood, the functional significance and location of the intravascular TF is unknown. In the plasma portion of blood, we found TF to be mainly associated with circulating microvesicles. By cell sorting with the specific marker CD42b, platelet‐derived microvesicles were identified as a major location of the plasma TF. This was confirmed by the presence of full‐length TF in microvesicles acutely shedded from the activated platelets. TF was observed to be stored in the α‐granules and the open canalicular system of resting platelets and to be exposed on the cell surface after platelet activation. Functional competence of the blood‐based TF was enabled when the microvesicles and platelets adhered to neutrophils, as mediated by P‐selectin and neutrophil counterreceptor (PSGL‐1, CD18 integrins) interactions. Moreover, neutrophil‐secreted oxygen radical species supported the intravascular TF activity. The pools of platelet and microvesicle TF contributed additively and to a comparable extent to the overall blood TF activity, indicating a substantial participation of the microvesicle TF. Our results introduce a new concept of TF‐mediated coagulation crucially dependent on TF associated with microvesicles and activated platelets, which principally enables the entire coagulation system to proceed on a restricted cell surface.

The functional role of blood platelet components in angiogenesis

The process of neovascularization greatly depends on the induction of the angiogenic phenotype of endothelial cells that is strictly controlled by humoral factors as well as by cellular communications in the vascular system. Although blood platelets contain several secretable pro- and antiangiogenic components, their overall role in angiogenesis remains poorly understood. In a mouse model of hypoxia-induced retinal angiogenesis, the situation of thrombocytopenia as well as inhibition of platelet aggregation by a highly specific alphaIIbbeta3-integrin antagonist or acetyl salicylic acid (Aspirin) administration, respectively, resulted in about 35-50% reduction of retinal neovascularization, compatible with a significant contribution of blood platelets in angiogenesis. Platelet remnants and microvesicles were found at sites of angiogenic sprouts. In vitro isolated platelets incorporated in a fibrin gel induced capillary sprouting of microvascular endothelial cells. Similarly, platelet releasate elevated the permeability of confluent endothelial cell monolayers to the same extent as hypoxia did. Platelet-derived VEGF as well as butanol-extractable lipid mediators were identified as predominant activators of angiogenesis, particularly of microvascular endothelial cell proliferation and migration. In addition, a synergistic effect between platelet-derived VEGF and bFGF in capillary sprouting and endothelial cell proliferation was found. Based on this proangiogenic role of platelets in neovascularization, anti-platelet substances can be considered as potent inhibitors of angiogenesis.

A novel type of co-chaperone mediates transmembrane recruitment of DnaK-like chaperones to ribosomes.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ‐like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK‐like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ‐like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J‐domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co‐chaperone, mediating transmembrane recruitment of DnaK‐like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK‐like chaperones of the endoplasmic reticulum.

Gray platelet syndrome: selective α-granule deficiency and thrombocytopenia due to increased platelet turnover

SummaryClinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and α-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of β-thromboglobulin were significantly reduced (3%–7% of normal). Platelet count was decreased (33–150×109/l) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.

The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion

In the present study the ability of plasminogen activator inhibitor type‐1 (PAI‐1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin‐dependent adhesion in a static system, mediated by αIIbβ3 on platelets and αv‐integrins on different megakaryoblastic cell lines, even though they also expressed αIIbβ3. In a concentration‐dependent manner, active, but not latent or complexed, PAI‐1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti‐adhesive effect of active PAI‐1. The direct binding of vitronectin, but not of other matrix proteins, to integrin αIIbβ3 was blocked by active PAI‐1 in a purified system. Since activated platelets release active and latent PAI‐1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co‐distribution of vitronectin and PAI‐1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI‐1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti‐proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin–integrin adhesion system that is influenced by PAI‐1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

The platelet contacts during aggregation

SummaryAPD-stimulated and aggregated platelets show dense structures (DS) on their free surfaces and filament bridges within 40–50 nm wide spaces of contacts between aggregating platelets (bridge contacts). Within the bridge contacts tight contacts are observed. Adjacent to tight contacts plasmalemmal openings into a canalicular system are seen. Pits and the central ends of these membranes are coated as seen in serial sections. Cationized ferritin (CF) added before stimulation binds to the whole negatively charged surface. Closer packed CF particles were observed on the DS and in the contacts on the central part of the bridges. CF did not introduce qualitative changes to the formation of typical platelet contacts, in particular of the tight contacts. Surface bound CF was found in the adjacent plasmalemmal invaginations and in the canalicular endomembranes. This result suggests an endocytosis mechanism which cleans the platelets of surface material during formation of tight contacts in aggregates.

Ultrastructural studies in a particular case of congenital dyserythropoietic anemia (CDA).

SummaryA case of CDA was investigated electron microscopically which clinically and serologically has been classified as type II. However, we have found the ultrastructural criteria both of CDA type I (open interconnections between heterochromatin of interphase nucleus and cytoplasm as well as alterations of the heterochromatin and nuclear envelope) and of type II (characteristic marginal spaces and sequestrations in the cytoplasm and nuclear protrusions). Only nuclear bridges — light microscopical characteristics of the CDA type I — were not observed. It was impossible to classify our case of CDA into type I or II from ultrastructural aspects. Two hypothetical explanations of these phenomena are discussed.ZusammenfassungEin Fall von CDA mit den klinischen und serologischen Zeichen des Typs II der Erkrankung wurde elektronenmikroskopisch untersucht. Dabei fanden sich Kernveränderungen in Erythroblasten, die als typisch für den Typ I der CDA angesehen werden (direkte Verbindungen zwischen Karyo- und Zytoplasma am Interphasenkern, Veränderungen des Heterochromatins und der Kernhülle). Daneben waren die für den Typ II der CDA charakteristischen Spalträume und Sequester im Zytoplasma erythropoietischer Zellen zu beobachten. Lediglich Kernbrücken — licht-mikroskopische Kennzeichen des Typs I — fehlten. Die Zuordnung des Bildes zu einem bestimmten Typ der CDA war auf Grund der morphologischen Kriterien nicht möglich. Obwohl fast alle unreifen Zellen und auch die Erythrozyten des peripheren Blutes alteriert waren, scheinen sie viele ihrer Funktionen wahrnehmen zu können.

The origin of the membrane convolute in degranulating platelets: a comparative study of normal and gray platelets

SummaryThrombin-stimulated normal platelets contain a membrane system of dilated channels with openings to the exterior. Whether these membranes originate from the surface connected system (SCS), the α-granules or internalized portions of the plasmalemma has not yet been defined. The present study traces in series of ultrathin sections the rearrangement of these membranes during shape change, degranulation and internalization of surface membranes in washed normal and “gray” platelets upon the stimulation with thrombin (1 IU/ml). Cationized ferritin (CF) was used as a surface marker in order to recognize internalized portions of the plasmalemma. Within the first seconds after stimulation, both normal and gray platelets changed their shape by extrusion of the SCS membranes. Simultaneously they started to internalize surface membrane and formed surface membrane invaginations closely attached to the outer rim of the cytoskeletal sphere which developed during the internal contraction. CF was internalized in these invaginations. CF was not observed within the system of dilated channels of stimulated platelets, however. Thrombin-stimulated gray platelets showed a markedly reduced number of dilated channels or none at all. This observation may be due to the fact “gray” platelets are deficient in α-granules. It is concluded that the dilated system of membranes in degranulated normal platelets originates from membranes of the α-granules which have performed compound exocytosis.

A New Model for in Vitro Clot Formation that Considers the Mode of the Fibrin(ogen) Contacts to Platelets and the Arrangement of the Platelet Cytoskeleton

Abstract: The constitution of platelet‐fibrin(ogen) contacts, the separation of the platelets initially aggregated, and the rearrangement of the platelet cytoskeleton during clot formation (0.5 to 60 minutes after thrombin stimulation) were investigated using ultrastructural and immunocytochemical techniques. After aggregation, fibrin polymerizing within focal contacts and from degranulating secretory granules contributed to the fibers. The initially formed focal contacts with fibrin obviously persisted during clot formation. The physiological branching of the fibers enabled separation of platelets. The contact associated cytoskeleton formed a constricting and fiber internalizing sphere, but later stress fiber like bundles. As retraction progressed, the cytoskeleton changed to stress fiber connecting focal contacts with fibers. A model of clot formation in vitro is presented that reflects both the contributions of platelets (fibrin fiber internalization and retraction) and of fibers (branching) enabling the retraction.