Biomedical subjects

E M Cramer

Publications and source records attributed to E M Cramer.

At least 19 recordsLinked to original sources

PubMed1996

Alpha-granule membrane mirrors the platelet plasma membrane and contains the glycoproteins Ib, IX, and V.

We have recently shown that several components from the platelet plasma membrane were also present at different rates in the alpha-granule membrane. This is the case for the glycoprotein (GP) IIb-IIIa (CD41), CD36, CD9, PECAM1, and Rap1b, while the GPIB-IX-V complex was considered to escape the rule. In this investigation, we studied the subcellular localization of GPIb, GPIX, and GPV in the resting platelets of normal subjects, patients with Bernard-Soulier syndrome, patients with Gray platelet syndrome, and human cultured megakaryocytes. Ultra-thin sections of the cells were labeled with antibodies directed against glycocalicin, GPIb, GPIX, and GPV. We have shown that a significant and reproducible labeling for the three GPs was associated with the alpha-granule membrane, accounting for approximately 10% of the total labeling. Furthermore, GPIb labeling appears Willebrand factor (vWF). After thrombin activation, vWF remained close to the limiting membrane of the open canalicular system (OCS), suggesting an early association of both receptor and ligand. Plasma membrane and alpha-granule labeling was virtually absent from the Bernard-Soulier platelets (characterized by a GPIb deficiency), thus proving the specificity of the reaction. In Gray platelets (storage granule deficiency syndrome), the small residual alpha-granules were also occasionally labeled for GPIb, GPIX, and GPIX. Cultured megakaryocytes that displayed the classical GPIb distribution, eg, demarcation and plasma membranes, exhibited also a discrete labeling associated to the alpha-granules. In conclusion, this study shows that, evenly for these three GPs, the alpha-granule membrane mirrors the plasma membrane composition. This might occur through an endocytotic process affecting each plasma membrane protein to a different extent and could have a physiologic relevance in further presentation of a receptor bound to its alpha-granule ligand to the platelet surface.

Bernard-Soulier Syndrome↗

PubMed1995

Absence of incorporation of plasma von Willebrand factor into porcine platelet alpha-granules.

In order to study the relationship between plasma and platelet von Willebrand factor (vWF), we used an experimental model of crossed bone marrow transplantation (BMT) between SLA immunocompatible normal and homozygous von Willebrand (vWD) pigs. A normal pig received bone marrow from a vWD pig and a second pig with vWD was engrafted with marrow from a normal pig. Each recipient, after total irradiation of 10 Grays, received by a central catheter 10(10) monocellular bone marrow cells without immunosuppression. The animals were followed for 50 d and no graft rejection or graft-versus-host disease was observed. After aplasia occurring 3 weeks after BMT, white blood cells and platelets returned to normal. Before transplantation, in the vWD pig, vWFAg and vWF activity were not detected in plasma and in platelet and megakaryocyte alpha-granules. After transplantation with normal marrow, platelet vWFAg and platelet vWF activity wer normal and high molecular weight multimers and numerous tubular structures were present in alpha-granules. Before transplantation, the normal pig had normal plasma and platelet vWFAg-vWF activity, normal multimeric pattern, and the platelet and megakaryocyte alpha-granules displayed many tubular structures, eccentrically located in one of their poles, coinciding with immunogold staining vWFAg. After transplantation with homozygous vWD marrow, platelet and megakaryocyte alpha-granules lacked tubular structures. Alpha-granule immunogold staining for vWF was consistently negative, although plasma vWF was at a normal level. In conclusion, this study shows that, unlike other plasma proteins such as fibrinogen. vWF endocytosis does not occur from plasma to the platelet alpha-granules. Platelet and megakaryocyte vWF solely originates from megakaryocyte endogenous synthesis and is independent of plasma vWF.

Animals↗

PubMed1994

Platelet alpha-granule and plasma membrane share two new components: CD9 and PECAM-1.

CD9 (p24) and PECAM1 (CD31) antigens are well-defined components of the platelet plasma membrane. Both are integral glycoproteins (GPs) implicated in the adhesive and aggregative properties of human platelets. In the present report, we have investigated their subcellular localization using immunoelectron microscopy. The monospecificity of the two polyclonal antibodies used was confirmed by immunoblotting. On normal resting platelets, immunolabeling for CD9 and PECAM1 was found lining the plasma membrane and the luminal face of the open canalicular system. Some labeling was also consistently found on the alpha-granule limiting membrane. This was confirmed by double labeling experiments in which fibrinogen and von Willebrand factor (vWF) were used as alpha-granule markers. CD9 and PECAM-1 were found lining the membrane of the same granules that contained fibrinogen and vWF in their matrix. CD9 and PECAM-1 thus appear to have an intracellular distribution identical to GPIIb-IIIa, a major aggregation platelet receptor. To rule out a cross-reactivity of the two polyclonal antibodies with GPIIb/IIIa, we studied PECAM1 and CD9 expression on the platelets from a patient with type I Glanzmann's thrombasthenia whose platelets are devoid of GPIIb/IIIa. The same pattern of labeling was observed for both antigens as for normal platelets. Normal platelets were further observed after stimulation by agonists that either fail to induce (ADP) or induce granule secretion (thrombin). After treatment with ADP, platelets changed shape and centralized their granules; the plasma membrane immunolabeling remained unchanged; and gold particles were still found decorating the periphery of the centralized alpha-granules. After thrombin treatment, alpha-granules fused with the platelet membrane and secretion occurred. A significant increase of labeling was then observed on the platelet surface. From these results we conclude that the alpha-granule membrane contains two additional receptors in common with the plasma membrane. This suggests that alpha-granule membrane receptors may originate from a dual mechanism: direct targeting from the Golgi complex in megakaryocytes (for alpha-granule-specific receptors such as P-selectin) or by endocytosis from the plasma membrane (for proteins distributed in the two compartments).

Adenosine Diphosphate↗

PubMed1994

Ultrastructural localization of the small GTP-binding protein Rap1 in human platelets and megakaryocytes.

Several functions have been proposed for Rap1B in human platelets, including the regulation of phospholipase (PL) C gamma and Ca2+ ATPase. However, its localization is largely unknown. In the present study we have investigated the subcellular distribution of Rap1 by immunocytochemical techniques using affinity purified polyclonal antibodies raised against residues 121-137 common to the 95% homologous Rap1A and Rap1B proteins. By immunofluorescence, a positive labelling was obtained on intact resting platelets and was abolished after adsorption of the antibodies with the control peptide. Immunoelectron microscopy was then used to further define the subcellular localization of Rap1B in platelets and megakaryocytes (MK). In resting cells, immunolabelling for Rap1B was associated with the plasma membrane, mostly at its inner face, and lined the membrane of the open canalicular system (OCS). Some labelling was also found outlining the alpha-granules, identified as such by a double labelling with an anti-GPIIb-IIIa. On thrombasthenic platelets the same localization was observed. When platelets were stimulated by thrombin, immunolabelling for Rap1B was redistributed to the zones of fusion of the granules with the OCS, and to the plasma membrane with a higher concentration on pseudopods. Human MK expressed Rap1 and the staining revealed the association of the protein with the demarcation membranes and alpha-granules. This study presents a first approach to the localization of a small GTP binding-protein Rap1B in whole platelets and MK, and shows its association with both the plasma and OCS membranes, as well as with the alpha-granule membranes.

Adenosine Diphosphate↗

PubMed1993

Ultrastructural demonstration of CD36 in the alpha-granule membrane of human platelets and megakaryocytes.

CD36 (glycoprotein [GP] IV) is a membrane GP of 88 kD found on monocytes, endothelial cells, and platelets. It may serve as a receptor for collagen and is also able to bind thrombospondin (TSP), because a monoclonal antibody to CD36 inhibits TSP binding to thrombin-stimulated platelets. In the following study, we investigated the subcellular distribution of CD36 within normal resting platelets, thrombin-stimulated platelets, and in cultured megakaryocytes (MK) by an immunogold staining technique and electron microscopy. We used an affinity-purified monospecific polyclonal antibody showing a single major band of precipitation at 88 kD via immunoblot analysis. In normal platelets, ultrastructural observation detected immunolabeling for CD36, homogeneously distributed along the platelet plasma membrane and in the luminal side of the open canalicular system (OCS). Moreover, some labeling was found around the alpha-granules along the inner face of their limiting membrane. An average of 70% of granules were labeled. The granule-associated pool of CD36 was estimated at approximately 25% of the total cell content. To exclude the possibility of a cross-reaction with GPIIb-IIIa, platelets from a patient with type I Glanzmann's thrombasthenia (which completely lack GPIIb-IIIa) were studied and showed a similar subcellular distribution of CD36, including alpha-granule membrane labeling. In activated platelets, CD36 was shown to be redistributed to the OCS and pseudopods of the plasma membrane. Platelets from a patient with the Gray platelet syndrome expressed CD36 on their plasma membrane, and some immunolabeling was also found within small abnormal alpha-granules. In cultured MK, CD36 immunolabeling was detected in the Golgi saccules, associated vesicles, immature alpha-granules, and demarcation membranes. In conclusion, this study shows the existence of a significant intragranular pool of CD36 in platelets that may play a critical role in the surface expression of alpha-granule TSP during platelet activation.

Adult↗

PubMed1993

Platelet alpha-granules.

Platelets contain a vast number of biologically active molecules within cytoplasmic granules which are classified according to their respective distinct ultrastructures, densities and content. The alpha-granule is a unique secretory organelle in that it exhibits further compartmentalization and acquires its protein content via two distinct mechanisms: (1) biosynthesis predominantly at the megakaryocyte (MK) level (with some vestigial platelet synthesis) (e.g. platelet factor 4) and (2) endocytosis and pinocytosis at both the MK and circulating platelet levels (e.g. fibrinogen (Fg) and IgG). The currently known list of alpha-granular proteins continues to enlarge and includes many adhesive proteins (e.g. Fg, von Willebrand factor (vWf) and thrombospodin (TSP)), plasma proteins (e.g. IgG and albumin), cellular mitogens (e.g. platelet derived growth factor and TGF beta), coagulation factors (e.g. factor V) and protease inhibitors (e.g. alpha 2-macroglobulin and alpha 2-antiplasmin). More recently the inner lining of the alpha-granule unit membrane has been demonstrated to contain a number of physiologically important receptors including glycoprotein IIb/IIIa (alpha IIb beta 3) and P-selectin. The alpha-granules originate from small precursor granules which can be observed budding from the trans-Golgi network within the platelet precursor cell the MK. During MK maturation the alpha-granules become very prominent and are ultimately packaged into platelets during thrombopoiesis. The alpha-granular contents are destined for release during platelet activation at sites of vessel wall injury and thus play an important role in haemostasis, inflammation, ultimate wound repair and in the pathogenesis of atherosclerosis.

Animals↗

PubMed1993

Porcine platelets contain an increased quantity of ultra-high molecular weight von Willebrand factor and numerous alpha-granular tubular structures.

Immunoelectronmicroscopy of human platelet alpha-granules reveals that von Willebrand factor (vWf:Ag) colocalizes with a small number of discrete tubular structures which appear identical to those observed within the Weibel-Palade bodies of endothelial cells. Although it is likely that tubules are composed of vWf:Ag as they are absent in severe vWD porcine platelets, their exact structural and functional nature is still unclear. In this study quantitative/qualitative analysis of vWf:Ag was undertaken in a series of platelet preparations obtained from normal pigs, normal humans and various vWD patients. Electron microscopy confirmed that normal pig platelet alpha-granules contain numerous, regularly spaced tubular structures eccentrically located and coincident with immunogold staining of vWf:Ag. In contrast, normal human platelet alpha-granules contain significantly fewer tubules (usually four to six) which are absent or reduced in number within various vWD platelet sections. Furthermore, the pig platelet lysates not only contained a full complement of multimers but also demonstrated significant intense staining of ultra-high MW material, irrespective of the presence or absence of proteolytic inhibitors. This ultra-high MW vWf appears similar to that observed within lysates prepared from endothelial cells and is susceptible to degradation to lower MW multimers. This study suggests that the tubular structures within alpha-granules and Weibel-Palade bodies may be composed of, or structurally related to, the ultra-high MW intracellular form of vWf:Ag.

Animals↗

PubMed1993

Reversibility of thrombin-induced decrease in platelet glycoprotein Ib function.

Thrombin induces a redistribution of glycoprotein (GP) Ib/GP IX complex from the platelet surface into the surface connected canalicular system (SCCS). This redistribution results in a reduced interaction of platelet GP Ib with von Willebrand factor (vWF) bound to subendothelium leading to impaired platelet adhesion. In this study we show that the platelet aggregation and degranulation require concentrations of thrombin above 0.05 U/ml, while the decrease in GP Ib function (about 50% of control value), as determined by ristocetin induced platelet agglutination, can be induced by lower concentrations (0.01-0.04 U/ml). Moreover, we show that when adding thrombin inhibitors to the platelets preincubated with < 0.04 U/ml thrombin for 5 min, their agglutinability by ristocetin was gradually recovered within 30 min, indicating that in these conditions the decrease in platelet adhesiveness is reversible. Immuno-electromicroscopic study showed that this restoration of platelet GP Ib function was associated with a reversed translocation of GP Ib from the SCCS to the plasma membrane. The data obtained from counting gold particles showed that the ratio of GP Ib immunolabelling on the external membrane versus that on the SCCS was 3.31 +/- 0.90 for resting platelets, down-regulated to 0.84 +/- 0.13 (P < 0.05 versus resting platelets) for the platelets treated with 0.04 U/ml thrombin and returned to 2.63 +/- 2.21 (P > 0.05 versus resting platelets) after incubation for 30 min with hirudin. However, the translocation of GP Ib was poorly reversed by thrombin inhibitors when higher concentrations of thrombin were used which induced platelet aggregation and large extent of degranulation. We conclude that thrombin affects platelets in a dose dependent manner, and that at low concentrations the decrease in platelet GP Ib related function is a reversible phenomenon.

Antithrombins↗

PubMed1993

Reversible translocation of glycoprotein Ib in plasmin-treated platelets: consequences for platelet function.

Understanding the effect of fibrinolysis on platelet function is of clinical importance. Plasmin is recognized to affect platelet adhesive function by reducing the interaction of platelet glycoprotein (GP) Ib with von Willebrand factor (vWF) bound to the subendothelium. This platelet function is commonly explored in vitro by the ristocetin-induced agglutination test. Our previous study demonstrated a plasmin-induced redistribution of GP Ib molecules from the platelet surface to the linings of the surface-connected canalicular system (SCCS), a critical mechanism for understanding plasmin-induced GP Ib dysfunction. Here, we demonstrate that neutralization of plasmin by its inhibitors, aprotinin or tripeptide Val-Phe-Lys-CH2Cl, permits a time dependent recovery (within 30 min) of ristocetin-induced agglutination in the platelets which were stimulated by plasmin at < 1 CU ml-1. This functional recovery was accompanied with a restoration of a normal amount of GP Ib on the platelet surface, as measured by the binding of both monoclonal anti-GP Ib antibody SZ 2 and 125I-labelled vWF to the platelets. Cytochalasin D did not inhibit this recovery, suggesting that this process may be due to passive actin depolymerization. These findings were further confirmed by immunoelectron microscopic study. Utilizing the platelets pre-labelled with anti-GP Ib antibody prior to plasmin stimulation, it was demonstrated that the observed recovery is due to a reverse translocation from the SCCS to the plasma membrane of the same GP Ib molecules which were present initially at the cell surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Agglutination↗

PubMed1993

Circulating megakaryocytes: delivery of large numbers of intact, mature megakaryocytes to the lungs.

To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 x 10(6) intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 x 10(6) partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.

Adult↗

PubMed1993

Gray platelet syndrome. Dissociation between abnormal sorting in megakaryocyte alpha-granules and normal sorting in Weibel-Palade bodies of endothelial cells.

The gray platelet syndrome (GPS) is a rare congenital bleeding disorder in which megakaryocytes and platelets are deficient in alpha-granule secretory proteins. Since the Weibel-Palade bodies (WPB) of endothelial cells as well as the alpha-granules contain the von Willebrand Factor (vWF) and P-selectin, we examined by transmission electron microscopy the dermis capillary network of two patients with GPS. Endothelial cells showed the presence of normal WPB with typical internal tubules. Using single and double immunogold labeling for vWF and P-selectin, we detected vWF within WPB, where it was codistributed with the tubules, whereas P-selectin delineated the outline of WPB. Therefore, the fundamental targeting defect in GPS is specific to the megakaryocytic cell line.

Biopsy↗

PubMed1993

[Expression of cyclin B in megakaryocytes and cells of other hematopoietic lines].

Megakaryocytes are normal bone marrow cells which have the unique ability to become polyploid. This phenomenon is termed endomitosis and its mechanism remains poorly understood at present. It is known that the cell cycle of eukaryotes, particularly at G2/M transition, is regulated by a complex with histone H1 kinase activity, the maturation- or M-phase promoting factor (MPF). We have therefore studied the expression of subunits of MPF, the p34cdc2 and cyclin B in normal bone marrow culture megakaryocytic cells, blood leukocytes and platelets as well as in human megakaryoblastic cell lines Dami, Meg-01, HEL and the promyelocytic cell line HL60. Using immunohistochemistry and electron microscopy we have observed that cyclin B was virtually undetectable in megakaryocytes and platelets, but was abundant in granulocytes, monocytes/macrophages and HL60. Studies by RT-PCR showed the presence in large quantities of mRNA of cyclin B in all cell types studied, even in megakaryocytic-like cells. These observations suggest some important implications in the understanding of the mechanisms of megakaryocyte polyploidization and related endomitosis.

Blood Platelets↗

PubMed1993

Effect of thrombin on maturing human megakaryocytes.

Thrombin causes platelet activation and secretion. In some nucleated cells, it is mitogenic. In this study, we have investigated how human megakaryocytes (MKs) respond to this agonist and whether the response depends on the maturation stage. MKs were cultured from bone marrow precursors in liquid culture in the presence of normal plasma. To determine whether thrombin can activate MKs, 14-day MK cultures were incubated with thrombin for 5 minutes, and cells were studied by electron microscopy, either by standard techniques or after embedding in glycol-methacrylate for immunoelectron microscopy. Ultrastructural examination of thrombin-treated MKs revealed dramatic morphological changes reminiscent of those found in platelets, including shape change and organelle centralization that involved immature as well as mature cells. MKs were also able to secrete alpha-granule proteins in the dilated cisternae of the demarcation membrane system, as shown by immunogold staining for thrombospondin and glycoprotein Ib. These changes were rapid (less than 5 minutes) but despite them, MKs remained viable for more than 24 hours. To determine whether thrombin has a mitogenic activity, it was added to the culture of MKs from day 3 to day 10 of culture at concentrations varying from 0.1 to 10 U/ml. Cells were subsequently studied by a double staining technique using flow cytometry to determine MK number and ploidy. No changes were observed in these two parameters, showing that thrombin is not mitogenic for MKs at the concentrations used. In conclusion, this study confirms for human MKs previous observations made about guinea pig MKs (Fedorko et al, Lab Invest 1977, 36:32). In addition, it demonstrates that immature MKs are able to respond to thrombin and that more mature cells can secrete alpha-granule proteins into the demarcation membrane system, which is in continuity with the extracellular space. This phenomenon may have implications for pathological states such as myelofibrosis formation and for megakaryopoiesis autocrine regulation.

Cell Count↗

PubMed1993

von Willebrand factor storage requires intact prosequence cleavage site.

Large multimers of the adhesive glycoprotein von Willebrand factor (vWf) are stored in endothelial cells in rod-shaped granules called Weibel-Palade bodies, while small multimers are secreted constitutively. Expression of pro-vWf in other cells with a regulated pathway of secretion, results in formation of vWf-containing storage granules that have a morphology similar to Weibel-Palade bodies. vWf expressed without its prosequence is not stored. To evaluate the importance of prosequence cleavage in vWf storage, the Arg at position -1, known to be necessary for cleavage, was mutated to Gly. Transfection of this cleavage mutant into two cell lines with a regulated pathway of secretion (RIN 5F and AtT-20 cells) led to the formation of large multimers. However, treatment of the cell lysates by the enzyme endoglycosidase H (Endo-H) did not reveal significant amounts of intracellular Endo-H-resistant vWf, which indicates the absence of a pool of stored processed vWf. In addition, no Weibel-Palade body-like structure was detected in these cells by immunofluorescence labeling with anti-vWf antiserum. Electron microscopy and immunocytochemistry of RIN 5F cells expressing the pro-vWf mutant confirmed the absence of Weibel-Palade body-like structures. In addition, anti-vWf-linked gold particles were found in the ER, occasionally in rounded granules and particularly in lysosomal structures which were abundant. We conclude that the formation of large aggregates is not sufficient to induce efficient vWf storage, and that the lack of cleavage of the prosequence may direct the mutant pro-vWf molecule to a degradative pathway. Therefore, the prosequence cleavage is a requirement for vWf storage.

Animals↗

PubMed1992

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.

Blood Platelets↗

PubMed1992

P-selectin, a granule membrane protein of platelets and endothelial cells, follows the regulated secretory pathway in AtT-20 cells.

P-selectin (PADGEM, GMP-140, CD62) is a transmembrane protein specific to alpha granules of platelets and Weibel-Palade bodies of endotheial cells. Upon stimulation of these cells, P-selectin is translocated to the plasma membrane where it functions as a receptor for monocytes and neutrophils. To investigate whether the mechanism of targeting of P-selectin to granules is specific for megakaryocytes and endothelial cells and/or dependent on von Willebrand factor, a soluble adhesive protein that is stored in the same granules, we have expressed the cDNA for P-selectin in AtT-20 cells. AtT-20 cells are a mouse pituitary cell line that can store proteins in a regulated fashion. By double-label immunofluorescence, P-selectin was visible as a punctate pattern at the tips of cell processes. This pattern closely resembled the localization of ACTH, the endogenous hormone produced and stored by the AtT-20 cells. Fractionation of the transfected cells resulted in the codistribution of P-selectin and ACTH in cellular compartments of the same density. Immunoelectron microscopy using a polyclonal anti-P-selectin antibody demonstrated immunogold localization in dense granules, morphologically indistinguishable from the ACTH granules. Binding experiments with radiolabeled monoclonal antibody to P-selectin indicated that there was also surface expression of P-selectin on the AtT-20 cells. After stimulation with the secretagogue 8-Bromo-cAMP the surface expression increased twofold, concomitant with the release of ACTH. In contrast, the surface expression of P-selectin transfected into CHO cells, which do not have a regulated pathway of secretion, did not change with 8-Br-cAMP treatment. In conclusion, we provide evidence for the regulated secretion of a transmembrane protein (P-selectin) in a heterologous cell line, which indicates that P-selectin contains an independent sorting signal directing it to storage granules.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Temperature dependence of plasmin-induced activation or inhibition of human platelets.

It is known that at 37 degrees C plasmin may have two opposite effects on platelets: at high concentrations (greater than 1.5 caseinolytic units [CU]/mL), plasmin activates platelets; at lower concentrations (0.1 to 1.0 CU/mL) it inhibits platelet activation induced by thrombin, collagen, or calcium ionophore A23187. In this study, we report that when lowering the incubation temperature to 22 degrees C, plasmin at low concentrations (0.1 to 0.5 CU/mL) fully activated platelets. When platelets were treated with 0.2 CU/mL of plasmin, lowering the incubation temperature from 37 degrees C to 22 degrees C resulted in an increase in the expression of fibrinogen receptors, in platelet release and aggregation. Thromboxane A2 was not generated by plasmin treatment at either temperature. Ultrastructural studies showed that platelets responded to low-dose plasmin at 37 degrees C by forming pseudopods, centralizing granules without fibrinogen release, whereas at 22 degrees C the same dose of plasmin caused platelet degranulation with the appearance of alpha-granule fibrinogen within the lumen of the surface connected canalicular system. In addition, at 22 degrees C plasmin at doses insufficient to induce platelet aggregation potentiated platelet response to thrombin. Thus, we suggest that plasmin may initiate both activating and inhibitory processes within platelets and that the change of temperature could influence this balance. These results may be of clinical relevance, because the fibrinolytic system was found activated during cardiopulmonary bypass in which the temperature of patient's blood circulation was reduced. This temperature-dependent behavior is also an interesting model for a further study on platelet response to serine proteinases.

Antibodies, Monoclonal↗

PubMed1991

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.

Blood Platelets↗