W. Jean Dodds

[4] Isolation of platelets from laboratory animals

Publisher Summary Known procedures for the separation of human platelets from peripheral blood are frequently adapted with variable success for the isolation of platelets from laboratory animal species. This chapter describes the process of isolating platelets from laboratory animals. Successful platelet isolation depends on the quality of the blood sample, which is influenced by the collection technique, the general health of the animal, and the uniformity of the subgroup or strain used. The techniques required to isolate platelets from the peripheral blood of all seven species are similar. Only blood samples obtained using a two-syringe technique and with minimal tissue trauma are suitable for platelet preparation. Platelet-rich plasma from all seven species can be used to obtain gel-filtered platelets (GFPs). The platelets are separated on a 1.5 × 30-cm column packed with acetone-washed Sepharose 4B containing 0.35% of bovine albumin. Following filtration, the GFP preparation is made 1 mM in CaCl. Under these conditions, more than 85% of platelet recovery can be expected from platelet-rich plasma (PRP). Human or species-specific fibrinogen is added to the GFP in most aggregation studies.

Ristocetin cofactor activity of purified canine factor VIII: inhibition by plasma proteins.

Abstract Canine plasma does not support the ristocetin-induced agglutination of fixed washed human platelets (FWP) under conditions that readily produce agglutination with human plasma. Purified canine factor VIII (FVIII) resembles purified human FVIII as it supported a pronounced ristocetin-induced agglutination of FWP in a system essentially free of extraneous proteins. Susceptibilities of these ristocetin cofactor activities (RCoF) to inhibition by plasmas or bovine serum albumin (BSA) were compared. Canine plasma, canine or human von Willebrands disease (VWD) plasmas, and BSA completely inhibited the RCoF of purified canine FVIII at concentrations that had minimal effects on this activity of purified human FVIII.

Impaired cAMP metabolism associated with abnormal function of thrombopathic canine platelets

The effects of forskolin and 1-Methyl-3-isobutyl-xanthine on thrombopathic platelet function and cyclic AMP accumulation were examined. The concentration of forskolin required to inhibit gamma-thrombin- induced platelet aggregation and secretion by 50% was significantly lower for thrombopathic than for normal platelets. This inhibition was accompanied by a marked elevation of cyclic AMP. 1-Methyl-3-isobutyl-xanthine, alone or in combination with forskolin, augmented both the cyclic AMP accumulation and the inhibition of platelet function. These results demonstrate that cyclic AMP metabolism is abnormal in thrombopathic platelets and imply that cyclic AMP-phosphodiesterase activity is impaired.

Hereditary and Acquired Thrombopathias

Normal platelet function is essential for hemostasis. The pathophysiologic processes that result in acquired quantitative platelet abnormalities are detailed. Platelet pathophysiology associated with acquired qualitative disorders, either drug-induced or secondary to disease, is discussed. Numerous hereditary platelet defects also exist. Their functional and biochemical features are presented.

Binding of Low-Molecular-Weight Canine Factor VIII Coagulant from von Willebrand Plasma to Canine Factor VIII-Related Antigen

Summary. Plasmas having no detectable factor VIII‐related antigen but moderate factor VIII coagulant were obtained from two unrelated dogs homozygous for von Willebrands disease and with the severe clinical expression of the disease. When these plasmas were gel‐filtered in a buffer at physiologic ionic strength, the factor VIII coagulant eluted in the bed volume as a single well‐defined peak. Addition of protease inhibitors, including diisopropylfluorophosphate, did not change the elution pattern. Each plasma was then combined individually with plasmas from six different mutants of canine haemophilia, all of which had normal factor VIII‐related antigen but no detectable factor VIII coagulant. The factor VIII coagulant elution profile of these combined plasma resembled that of normal canine plasma. Slightly over half of the recovered factor VIII coagulant coeluted in the void volume with the factor VIII‐related antigen; the rest eluted as a second, distinct peak of lower molecular weight. These results demonstrated that part of the factor VIII coagulant of the von Willebrand plasmas had bound to the factor VIII‐related antigen of the haemophilic plasmas. This finding supports the theory that factor VIII exists as a macromolecular complex of nonidentical components in normal citrated plasma.

Factor VIII-related antigen in canine hemophilia and von Willebrand's disease: Variation when measured with different agaroses☆

The source of agarose used in the Laurell electroimmunoassay was found to produce variations in the factor VIII-related antigen levels of plasmas from dogs with von Willebrands disease and hemophilia A. Similar artifactual variations in antigen were not observed for normal canine plasmas.

Physical Relationship between Canine Factor VIII Coagulant Activity and Factor VIII-Related Antigen

SummaryTwo of three insolubilized anti-canine FVIII systems removed similar amounts of VIII-C and VIII-RA from canine plasma. These data suggest that VIII-C and VIII-RA form part of one circulating complex.The authors thank Dr. W. P. Webster, Chapel Hill, N.C., for providing the canine hemophilia A plasma and Joanne E. Kull for her assistance in collecting canine plasma samples and immunizing the rabbits.Summary Two of three insolubilized anti-canine FVIII systems removed similar amounts of VIII-C and VIII-RA from canine plasma. These data suggest that VIII-C and VIII-RA form part of one circulating complex. The authors thank Dr. W. P. Webster, Chapel Hill, N.C., for providing the canine hemophilia A plasma and Joanne E. Kull for her assistance in collecting canine plasma samples and immunizing the rabbits.

Purified cobra venom factor: effect on blood platelets.

Summary A specific complement-reactive factor derived from cobra venom (CVF) inhibited complement activity, impaired clot retraction, and induced aggregation and release of intracellular constituents in plateletrich plasma or suspensions of washed platelets from guinea pigs, rabbits, dogs, and humans. Platelets from thrombopathic dogs and from a complement deficient (C3) patient responded similarly when mixed with purified CVF; possible explanations for these observations are included. We are indebted to Mr. Jack Newman, New York University Medical Center, for testing our CVF preparations for the presence of contaminating fibrinolytic enzymes.SummaryA specific complement-reactive factor derived from cobra venom (CVF) inhibited complement activity, impaired clot retraction, and induced aggregation and release of intracellular constituents in plateletrich plasma or suspensions of washed platelets from guinea pigs, rabbits, dogs, and humans. Platelets from thrombopathic dogs and from a complement deficient (C3) patient responded similarly when mixed with purified CVF; possible explanations for these observations are included.We are indebted to Mr. Jack Newman, New York University Medical Center, for testing our CVF preparations for the presence of contaminating fibrinolytic enzymes.

Complement C5a (desArg) generation in serum exposed to damaged aortic endothelium

Complement activation by injured endothelial cells was investigated using ex vivo rabbit thoracic aortas as a source of endothelium and a neutrophil aggregation (NA) bioassay to detect the complement cleavage product C5a (desArg). Endothelium, oxygen-starved by incubating aortas 30 min with Tyrodes buffer, activated complement as demonstrated by a 57% increase in the NA response induced by serum from buffer-treated aortas as compared to serum from untreated control aortas. Incubation of MgEGTA serum in injured aortas resulted in a 27% (P less than 0.025) weaker NA response than normal serum, indicating participation by both classical and alternative pathways of complement activation. Serum from aortas incubated 30 min with 100 micrograms/ml cholestane-3 beta, 5 alpha, 6 beta-triol, a cytotoxic cholesterol oxidation derivative, induced a NA response comparable to that from serum from aortas treated 30 min with Tyrodes buffer. Heat inactivation of serum prior to aortic incubation abolished NA activity and serum incubated in deendothelialized aortas lacked NA activity. Fractionation of serum samples from these experiments on Sephadex G-100 revealed a single peak of NA activity corresponding to the molecular weight of C5a (desArg). Endothelial cell injury was demonstrated by the inability to exclude Trypan blue dye and by scanning electron microscopy. These data demonstrate that damaged arterial endothelium can effectively activate the complement system, resulting in the production of an anaphylatoxic inflammatory mediator.Complement activation by injured endothelial cells was investigated using ex vivo rabbit thoracic aortas as a source of endothelium and a neutrophil aggregation (NA) bioassay to detect the complement cleavage product C5a (desArg). Endothelium, oxygen-starved by incubating aortas 30 min with Tyrodes buffer, activated complement as demonstrated by a 57% increase in the NA response induced by serum from buffer-treated aortas as compared to serum from untreated control aortas. Incubation of MgEGTA serum in injured aortas resulted in a 27% (P less than 0.025) weaker NA response than normal serum, indicating participation by both classical and alternative pathways of complement activation. Serum from aortas incubated 30 min with 100 micrograms/ml cholestane-3 beta, 5 alpha, 6 beta-triol, a cytotoxic cholesterol oxidation derivative, induced a NA response comparable to that from serum from aortas treated 30 min with Tyrodes buffer. Heat inactivation of serum prior to aortic incubation abolished NA activity and serum incubated in deendothelialized aortas lacked NA activity. Fractionation of serum samples from these experiments on Sephadex G-100 revealed a single peak of NA activity corresponding to the molecular weight of C5a (desArg). Endothelial cell injury was demonstrated by the inability to exclude Trypan blue dye and by scanning electron microscopy. These data demonstrate that damaged arterial endothelium can effectively activate the complement system, resulting in the production of an anaphylatoxic inflammatory mediator.

Animal Models for the Evolution of Thrombotic Diseasea

Naturally occurring hemorrhagic and thrombotic diseases of animals closely parallel their human counterparts. While such models may be particularly useful in studying the pathogenesis of human disease, it is usually more realistic to depend upon experimentally induced disease models. The species selected for use is therefore of major importance in providing meaningful extrapolation to humans, as are the experimental design and type of procedure (in vitro, ex vivo, in vivo). Regardless of the test system used when in vitro procedures are employed, these must be translated eventually to the in vivo situation. Information about the normal aging process of different species is important here and should influence selection of the species and test system. The ideal situation may not be feasible or pertain because of cost, availability, size, and investigator familiarity, or lack thereof, with the most suitable species or model.