The roles of ADP and TXA in botrocetin/VWF-induced aggregation of washed platelets.

BACKGROUND: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to alphaIIbbeta3 activation and platelet aggregation. The roles of ADP and thromboxane A2 (TXA2) in agglutination-induced GPIbalpha-mediated platelet activation have not been fully described. METHODS: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA2 receptors, Galphaq, or alphaIIbbeta3, and inhibitors and chelating agents were used to investigate the roles of TXA2, ADP, alphaIIbbeta3 and Ca2+ in botrocetin/VWF-induced signaling. RESULTS: Our data demonstrate that botrocetin/VWF/GPIbalpha-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIbalpha-elicited TXA2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA2-induced alphaIIbbeta3 activation and ADP signaling. TXA2 or ADP can activate alphaIIbbeta3, but both are required for alpha-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Galphaq-dependent. alpha-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an alphaIIbbeta3-dependent manner. CONCLUSIONS: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates alphaIIbbeta3 requires TXA2. Agglutination-elicited TXA2 production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes alphaIIbbeta3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.

AlphaIIbbeta3-mediated outside-in signaling induced by the agonist peptide LSARLAF utilizes ADP and thromboxane A2 receptors to cause alpha-granule secretion by platelets.

The peptide LSARLAF (LSA) causes alphaIIbbeta3-dependent platelet activation that results in alpha-granule secretion and aggregation. LSARLAF-induced, alphaIIbbeta3-mediated outside-in signaling causing alpha-granule secretion and platelet aggregation was studied using washed mouse platelets. ADP receptor antagonists, enzyme inhibitors, normal platelets and platelets from mice that lack either Galphaq or thromboxane (Tx) A2 receptors were used for this investigation. The results demonstrate that LSA-induced alphaIIbbeta3-mediated signaling producing aggregation of washed platelets is mediated through the release of ADP and thromboxane, which cause alpha-granule release by mediating their effects though Galphaq and/or Gi depending on the level of LSA used to activate the platelets. Specifically, alphaIIbbeta3 elicited aggregation of washed platelets in response to a low level of LSA requires signaling through the ADP receptor P2Y1 and Galphaq, and the ADP receptor P2Y12 and Gi as well as TxA2 receptors. However, this aggregation is independent of Galphaq and TxA2 signaling in response to high LSA concentrations, but is dependent on ADP signaling through its receptor P2Y12, and therefore presumably Gi, regardless of the level of LSA used to activate the platelets. PKC function is required for ADP secretion and the subsequent signaling through P2Y12 regardless of the level of LSA used to activate the platelets. The end point of the LSA-induced alphaIIbbeta3-mediated signaling characterized in this study is alpha-granule secretion, which provides the fibrinogen required for aggregation of washed platelets.

Prolonged bleeding time with defective platelet filopodia formation in the Wistar Furth rat.

Hereditary macrothrombocytopenia is a hallmark of Wistar Furth (WF) rats. In addition, a platelet/megakaryocyte alpha granule defect, similar to that of patients with gray platelet syndrome, is present. Several observations indicate cytoskeletal abnormalities in WF platelets and megakaryocytes, suggesting the potential for functional defects in hemostatic processes requiring cytoskeletal reorganization, such as platelet adhesion and spreading. However, no bleeding abnormality has been noted. Here, we report a prolonged bleeding time (>30 minutes in 10 of 11 rats tested) with defective clot formation in the WF strain. Prolonged bleeding time can result from defects in platelet adhesion, aggregation, or the release reaction. Because aggregation to collagen and adenosine diphosphate were reported to be normal, we determined whether WF rat platelets are defective in their ability to adhere to substrates. Platelet adherence and spreading was evaluated from 30 seconds to 30 minutes on Formvar-coated, carbon-stabilized grids or poly-L-lysine-coated glass coverslips by transmission electron microscopy or immunofluorescence, respectively, and scanning electron microscopy. We classified the adhered platelets according to their pattern of spreading, ie, rounded, rounded or spreading with short filopodia, spindle-shaped, spreading with long filopodia, spreading with lamellipodia, and fully spread. Adherent normal rat platelets displayed all stages of spreading within 30 seconds to 2 minutes, including many spindle-shaped forms, and forms with multiple, long filopodia. In contrast, adhered WF platelets at these early time points rarely developed long filopodia or were spindle shaped. The majority of adherent WF platelets at these early time points were either round, spread with a few short filopodia, or extensively spread with wide lamellipodial skirts. By 15 to 30 minutes, most platelets in both Wistar and WF samples were fully spread. These data show abnormal WF platelet spreading. The paucity of spindle-shaped forms and forms with long filopodia may reflect an inability of WF platelets to undergo the early stages of spreading, or, alternatively, their more rapid than normal progression through these stages. We hypothesize that this failure to spread normally may relate to prolonged bleeding times in vivo and defective clot formation in WF rats.

Identification of the Src family kinases, Lck and Fgr in platelets. Their tyrosine phosphorylation status and subcellular distribution compared with other Src family members.

We have identified the Src family members, Lck and Fgr in resting human and rodent platelets and compared their subcellular distributions and tyrosine phosphorylation status to those of the other Src family kinases to gain insights into the signal transduction pathways active in maintaining platelets in the circulation. Like Fyn, Lyn, and Yes, most of Fgr and Lck was detergent-insoluble in human and rat platelets. In comparison, Src showed higher detergent solubility than the Src-related kinases. Most all human platelet Src was detergent-soluble, while that of rodent platelets was present in all detergent fractions. We also compared the tyrosine-phosphorylation status of Lck and Fgr to other Src family members in resting platelets using immunoprecipitation and immunoblotting. All of these Src family members except Fgr exhibited substantial phosphotyrosine antibody labeling. The partitioning of these kinases, with the exception of Src, with the detergent-insoluble fraction, their tyrosine-phosphorylation status, and co-localization with endocytotic vesicles lead us to hypothesize that the Src family kinases are involved in signaling events that drive cytoskeletal reorganization and active endocytosis of plasma proteins by circulating platelets.

Megakaryocytopoiesis and platelet production are stimulated during late pregnancy and early postpartum in the rat.

Platelet count during uncomplicated pregnancy shows considerable patient variation. To gain a better understanding of thrombocytopoiesis during pregnancy, megakaryocytes and platelets were examined during gestation and the early postpartum period, using as a model the rat. Platelet counts and megakaryocyte concentrations and DNA content distributions of timed-pregnant rats were examined at intervals from day 10 of gestation through parturition on day 22 and days 1 through 7 postpartum. Platelet survival was studied in late gestation and the early postpartum. Platelet volume was measured on gestation day 21. Platelet counts were moderately increased on gestation days 17 and 19 through 22, and on days 2 to 3 postpartum. However, the actual rate of platelet production was much higher than the platelet count suggests because the blood volume increased in late gestation to 1.5 times the nonpregnant level. Mean platelet volume and platelet volume distribution width of day 21 gestation rats were not significantly altered. Platelet survival in pregnant rats was not significantly different from that in nonpregnant females. In contrast, megakaryocyte concentration was significantly increased on gestation days 12, 17, and 19 through 21, and 2 to 3 days postpartum. In addition, in late gestation, megakaryocyte DNA content distributions displayed a marked increase in the proportion of high ploidy cells, which peaked 1 day before parturition. At that time, the proportions of 32N (43%) and 64N cells (3%) were, respectively, three and four times nonpregnant values. In contrast to megakaryocyte concentration, megakaryocyte DNA content distributions had returned to the nonpregnant pattern by day 1 postpartum. The changes in megakaryocyte DNA content distribution were accompanied by changes in megakaryocyte size. These data indicate that thrombopoiesis is substantially increased during late pregnancy, and that this increase is accomplished through an increase in megakaryocyte DNA content and size, as well as megakaryocyte number. The more rapid return of megakaryocyte DNA content than of megakaryocyte concentration to nonpregnant levels postpartum suggests that pregnancy-associated hormonal changes which produce an increase in megakaryocyte DNA content and size differ from those which cause an increase in megakaryocyte number.

A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth rat.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.

Comparison of platelet production in two strains of mice with different modal megakaryocyte DNA ploidies after exposure to hypoxia.

Thrombocytopenia develops with prolonged exposure to hypoxia. Although decreases in megakaryocyte numbers due to hypoxia have been well documented, the effects of hypoxia on megakaryocyte DNA content have not been reported. In this study, megakaryocytopoiesis and platelet production were compared in both C3H mice (whose megakaryocyte modal ploidy class is 32N) and C57/BL mice (whose modal ploidy class is 16N), by enclosure in cages covered with silicone-rubber membranes. After equilibration, O2 levels inside the cages were 6%-7%. Hematocrits, platelet counts, platelet sizes, percent 35S incorporation into platelets, megakaryocyte size and number, and megakaryocyte DNA content of mice were measured before and at various days after hypoxia. Although hematocritis increased and platelet counts decreased in both strains of mice with time in hypoxic chambers, megakaryocyte and platelet responses of C3H mice differed from those of C57/BL mice in several respects; hematocrits of C3H mice were higher and platelet counts were lower than those in C57/BL mice. C3H mice produced larger platelets than C57/BL mice in response to hypoxia. Total circulating platelet counts (TCPC) and total circulating platelet masses (TCPM) of both mouse strains showed similar biphasic responses, that is, elevated TCPC and TCPM on days 2-4 and decreased values after 6-14 days of hypoxia. However, hypoxic C3H mice had lower TCPC on days 4-14 and lower TCPM on days 10-14 of hypoxia than C57/BL mice. Both C3H and C57/BL mice had decreased megakaryocyte numbers at 6-10 days of hypoxia, but only C3H mice had decreased numbers of megakaryocytes at day 14. Elevated megakaryocyte size was observed in both mouse strains at day 14 of hypoxia. However, after hypoxia, C3H mice showed a greater depression in megakaryocyte number and a larger increase in megakaryocyte sizes than did C57/BL mice. C3H mice maintained 32N as the modal megakaryocyte DNA content through day 10 of hypoxia, but 64N was the modal megakaryocyte DNA content at day 14; 16N remained the modal megakaryocyte DNA content in hypoxic C57/BL mice. Hypoxic C3H mice had an increase in 16N megakaryocytes after 6 days of hypoxia, followed by an increase in the proportion of 64N cells at 14 days compared to values of untreated C3H control mice. Hypoxic C57/BL mice had an increased proportion of 16N cells at 6 days but a decreased proportion of 32N cells at 14 days. These studies demonstrate that the decreased platelet production resulting from prolonged exposure to hypoxia is primarily the result of decreased differentiation of hematopoietic precursors into the megakaryocyte lineage rather than decreased megakaryocyte DNA content, because higher ploidy classes actually increase as thrombocytopenia becomes more severe. Stem cell competition could explain the findings of reduced platelet production and increased red blood cell production in both strains of mice after exposure to hypoxia.

Letters.

WHO HAS UPDATE ON RESEARCH EDUCATING DOCTORS A NATIONAL RESOURCE? A HOLISTIC APPROACH MORE SAMPLES NEEDED A USEFUL RESOURCE THE APPLIANCE OF SCIENCE WILL THE PRICE BE RIGHT? COMMUNITY NEEDS DISPARITY IN CARE BACK TO BASICS INFECTION MATTERS PREACHING TO THE CONVERTED? SUPPORT FOR STAFF A NURSE-LED DISCIPLINE INNOVATION NEEDED PRESCRIBING IMPLICATIONS.

Platelets of the Wistar Furth rat have reduced levels of alpha-granule proteins. An animal model resembling gray platelet syndrome.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia (large mean platelet volume [MPV] with increased platelet size heterogeneity and reduced platelet count). Ultrastructural studies suggest that this anomaly results from erratic subdivision of megakaryocyte cytoplasm into platelets. In this study, we have examined protein profiles of platelets of WF rats for biochemical abnormalities associated with this anomaly. Marked decreases in protein bands with an Mr of 185, 57, 53, 16, 13, and 8 kd were observed in one-dimensional reduced SDS-PAGE gels in WF platelets compared with platelets of Wistar, Long Evans, and Sprague-Dawley rats. These proteins were released into the supernatant when washed platelets were treated with thrombin suggesting that they were alpha-granule proteins. These abnormalities were not present in offspring of crosses between Wistar Furth and Wistar rats; however, they were present in platelets of offspring with large MPV derived from backcrosses of (WF X Wistar) F1 males to WF females, but not in backcross offspring with normal platelet size. Immunoblotting confirmed decreased levels of thrombospondin, fibrinogen, and platelet factor 4 in WF platelets. Electron microscopic examination revealed that platelet alpha granules were usually smaller in Wistar Furth than in Wistar rats. In addition, immunogold electron microscopy demonstrated that the surface connected canalicular system of the large Wistar Furth platelets, contained dense material composed of alpha-granule proteins, not present in Wistar platelets. From these results, we conclude that the Wistar Furth rat platelet phenotype of large mean platelet volume and decreased levels of alpha-granule proteins represents an animal model resembling gray platelet syndrome. The autosomal recessive pattern of inheritance of the large MPV phenotype and platelet alpha-granule protein deficiencies suggests that a component common to both formation of platelet alpha granules, and subdivision of megakaryocyte cytoplasm into platelets, is quantitatively or qualitatively abnormal in Wistar Furth rat megakaryocytes and platelets.

Prolonged thrombocytosis in mice after 5-fluorouracil results from failure to down-regulate megakaryocyte concentration. An experimental model that dissociates regulation of megakaryocyte size and DNA content from megakaryocyte concentration.

Rodents treated with 150 mg/kg of 5-fluorouracil (5-FU) exhibit a marked and prolonged rebound thrombocytosis, suggesting that feedback control of one or more megakaryocyte characteristics (size, polyploidy, or concentration) is altered. To determine the changes in megakaryocytes that lead to such a profound thrombocytosis, C3H mice were injected with 150 mg/kg 5-FU, and platelet and megakaryocyte responses were examined at frequent intervals from days 1 through 25. After 5-FU injection, all megakaryocyte indices decreased, as did platelet number. However, the decrease in platelets to one third of control was greater than the decreases in megakaryocyte indices, suggesting that thrombocytopoiesis was ineffective from days 3 through 7 post 5-FU. Megakaryocyte size began to recover on day 4, followed by polyploid DNA content on day 5, and megakaryocyte concentration and platelets at 7.5 days. Megakaryocyte size peaked on days 6 through 8 (1.25 x normal), followed by megakaryocyte polyploid DNA content on day 8, megakaryocyte concentration on days 9 through 12 (2 1/2 to 3x normal), and platelets on days 12 through 15 (2x normal). Platelet levels are thought to be important in the feedback regulation of megakaryocytes; however, only polyploid DNA content distributions showed a close inverse relationship to platelet counts during both the recovery and rebound thrombocytosis phases after 5-FU. In contrast, megakaryocyte size peaked before platelet recovery commenced, while megakaryocyte concentration increased in parallel with platelets from 7.5 to 10 days post 5-FU and continued to be maintained at 2 to 3 times normal through day 13, despite platelet levels that were more than twice normal. Both megakaryocyte size and polyploid DNA content distributions shifted toward lower values in response to the rebound thrombocytosis (DNA content on day 10 and size on days 12 and 13). Splenectomy did not substantially alter the pattern of post 5-FU rebound thrombocytosis or megakaryocyte response from that seen in intact mice, indicating that splenic megakaryocytes are not responsible for the prolonged thrombocytosis seen after this drug. In summary, the prolonged thrombocytosis after 5-FU administration results from failure to down-regulate the number of precursors entering the differentiating megakaryocyte compartment. These data indicate that megakaryocyte size and DNA content are responsive to different feedback controls than megakaryocyte concentration in this model system.

An analysis of megakaryocytopoiesis in the C3H mouse: an animal model whose megakaryocytes have 32N as the modal DNA class.

The modal DNA content of normal marrow megakaryocytes from species so far examined usually has been reported to be 16N. In this report we describe an exception in the C3H mouse whose megakaryocytes have a modal DNA content of 32N. Female C3H/HEN mice had an average DNA content distribution of 14% 8N, 37% 16N, 43% 32N, and 6% 64N. Male C3H/HEN mice had somewhat higher proportions of 32N and 64N megakaryocytes (average DNA content distribution of 12% 8N, 29% 16N, 47% 32N, and 12% 64N) than females. All 11 other mouse strains examined had 16N as the modal megakaryocyte DNA content, although the proportions in the various polyploid DNA classes showed some strain variation. Megakaryocyte size was similar among all 12 strains evaluated, and mean platelet volume (MPV) of C3H/HEN mice differed from only 1 of the other 4 strains analyzed. Platelet counts of C3H/HEN mice were similar to those of six, and slightly but significantly lower than those of five other mouse strains examined. Compared with megakaryocyte concentrations of other mouse strains studied, that of C3H/HEN mice was similar to seven, somewhat higher than one, and slightly lower than three strains. Offspring from reciprocal matings of C57BL/6 and C3H/HEN mice had megakaryocyte DNA distributions intermediate between those of the parent strains, suggesting that a higher gene dosage of some component is responsible for the right-shifted megakaryocyte DNA content distribution phenotype of C3H mice. The proportions of 32N and 64N megakaryocytes increased in C3H/HEN mice in response to acute thrombocytopenia, as did those of CBA/CAJ mice used as a comparative strain. In summary, megakaryocytes of the C3H mouse have a higher average DNA content but similar platelet count, MPV, and megakaryocyte size and concentration as those of most other mouse strains. These results suggest that the number of platelets produced per unit of C3H megakaryocyte DNA is less than that for other mice.

Genetic and physiological variations in megakaryocyte DNA content distributions.

The DNA content of normal megakaryocytes usually ranges from 8N to 64N, with 16N as the modal DNA content. The frequency of cells at each DNA content can be altered by experimental induction of thrombocytopenia, thrombocytosis or marrow ablation, and in various disease states; however, the mechanisms and regulation involved in the process of polyploidization remain obscure. This discussion will focus on genetic and physiologic variations in megakaryocyte DNA content distributions. The genetic variations are those we have observed among mouse strains, with the most pronounced present in several C3H substrains in which the modal megakaryocyte DNA content is 32N, rather than 16N. The physiologic variation reported here is a shift to the right in megakaryocyte DNA content distributions during late pregnancy in the rat.

The Wistar Furth rat: an animal model of hereditary macrothrombocytopenia.

The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.

Increase in circulating megakaryocyte growth-promoting activity (Meg-GPA) following sublethal irradiation is not related to decreased platelets.

In this study, we have used the sublethally irradiated rat as a model to examine the relationship between platelet count and the plasma level of megakaryocyte growth-promoting activity (Meg-GPA) assayed in vitro. Meg-GPA of irradiated rats whose platelet counts were maintained by platelet transfusions was compared to that of irradiated controls allowed to develop thrombocytopenia. The irradiated controls were given either saline, packed red cells, or no other treatment. Blood values were determined and plasma was collected from all groups 11 days after irradiation, predetermined to be the nadir of platelet count and the peak of Meg-GPA. The Meg-GPA of plasma was assayed by culturing in vitro at a final concentration of 30% with rat marrow in methylcellulose and 2-mercaptoethanol for 7 days at 37 degrees C. Plasma from platelet-transfused irradiated rats, when collected at ambient temperature, contained markedly decreased levels of Meg-GPA compared to that present in plasma of irradiated controls. Subsequent in vitro studies indicated that elevated Meg-GPA levels of plasma from irradiated thrombocytopenic rats were drastically reduced by incubation with platelets in vitro at 37 degrees C. Incubation of the same plasma with platelets at 4 degrees C resulted in much less reduction in Meg-GPA. This suggested that the diminished Meg-GPA observed with plasma from the platelet-transfused irradiated rats may have been due to activation and release of Meg-GPA inhibitors from platelets during plasma collection. To investigate this possibility, experiments were repeated in which platelets of irradiated rats were maintained by platelet transfusion, but plasma was collected at 4 degrees C. Under these conditions, the level of Meg-GPA in plasma of the platelet-transfused irradiated rats was not markedly different from that in plasma of irradiated controls. We conclude that in this experimental model, circulating Meg-GPA level is not related to platelet count.

Interaction of ristocetin and bovine plasma with guinea pig megakaryocytes: a means to enrich megakaryocytes based on membrane rather than physical characteristics.

We have investigated whether megakaryocytes can be aggregated by ristocetin and bovine plasma and whether such aggregation can be used as a step in the purification of megakaryocytes from marrow cell suspensions. Guinea pig marrow cell suspensions were first enriched for megakaryocytes by density equilibrium centrifugation in continuous Percoll density gradients. The megakaryocyte-enriched marrow was stirred in a platelet aggregometer to which ristocetin or bovine plasma was added. Megakaryocytes were aggregated by both ristocetin and bovine plasma with the proportion aggregated being related to the concentration of ristocetin or bovine plasma. Maximal aggregation (greater than 90% of megakaryocytes) was achieved with 2.0 mg/mL ristocetin or 5% bovine plasma and required five minutes. All maturation stages of morphologically recognizable megakaryocytes were aggregated. The megakaryocyte aggregates were separated from the marrow suspension by sedimentation at 1 g and the megakaryocytes disaggregated by dilution with media (ristocetin aggregated) or addition of dextran sulfate (bovine plasma aggregated). Megakaryocyte purity and recovery were higher with bovine plasma than with ristocetin. A mean of 92% of the megakaryocytes in the bovine plasma aggregated cell suspensions were recovered with megakaryocytes constituting an average of 76% of the final cell suspensions. The viability as well as the diameters and DNA content distribution of these megakaryocytes were similar to those of the starting population. We conclude that guinea pig megakaryocytes behave like platelets in that they can be aggregated with ristocetin or bovine plasma and that megakaryocyte aggregation induced by ristocetin or bovine plasma provides a means to enrich these cells based on membrane rather than physical characteristics. This approach yields purified megakaryocyte populations that are representative of those in unfractionated marrow.

Inverse relationship between megakaryocyte buoyant density and maturity.

We examined the relationship between rat megakaryocyte buoyant density and maturation stage in continuous Percoll density gradients. An average of 88% of megakaryocytes had buoyant densities less than 1.054 g/ml. There was an inverse relationship between megakaryocyte buoyant density and maturation. Morphologically mature forms comprised 90% of the megakaryocytes with buoyant densities of 1.030-1.033 g/ml. In contrast, immature morphology was present in three-quarters of megakaryocytes with buoyant densities of 1.042-1.046 g/ml. These morphological findings were confirmed by [3H]thymidine labelling studies. Cell viability assessed by trypan blue exclusion was highest among more dense megakaryocytes of which the majority were immature. The lowest trypan blue exclusion was found in the less dense, predominantly mature megakaryocytes indicating that these cells are more susceptible to membrane damage during marrow suspension. Megakaryocyte DNA content distributions and platelet antigen levels, determined by two-colour flow cytometry, were also related to megakaryocyte density; the more dense megakaryocytes showed an approximately two-fold higher proportion of 8N cells and less platelet antibody binding than did less dense megakaryocytes. These studies suggest that megakaryocytes can be fractionated according to their buoyant densities into immature and mature populations suitable for molecular studies of differentiation.