Kevin O. Garrett

Initial Platelet Deposition at the Human Microvascular Anastomosis: Effect on Downstream Platelet Deposition to Intact and Injured Vessels

Initial platelet deposition (PD) in and around the region of a small-vessel anastomosis may set the stage for thrombosis and tissue loss. To study this problem, a human vessel model (human placental artery, HPA) has been designed to mimic the vascular injuries attendant on clinical microsurgery. To perform these studies, dissected lengths of human placental artery were treated to provide the following four types of injury: group I: control, dissected but otherwise uninjured (N = 5); group II: distal portion of vessel endothelium removed (N = 5); group III: central anastomosis, distal endothelium intact (N = 7); and group IV: central anastomosis, distal endothelium removed (N = 4). Vessels were perfused with 25 ml human whole blood for 17 ± 5 s at an average shear rate of 536 s−1. Vessels in groups I to IV were segmented at 2-cm intervals, and the number of 111In-labeled plateles was measured. Data from the following groups of exposure zones were pooled and analyzed: endothelium intact, endothelium absent, anastomosis present, postanastomosis with endothelium intact, and postanastomosis with endothelium absent. Significant numbers of platelets were found to attach to intact endothelium, indicating that ischemia and micro-surgical handling may augment platelet deposition to otherwise uninjured vessels. A similar degree of platelet deposition was measured after exposure of the subendothelium and perfusion, indicating that superficial subendothelial exposure in the absence of an additional prothrombotic stimulus may lead to no greater platelet deposition than occurs on slightly injured endothelium alone. Platelet deposition at anastomoses was strikingly elevated, although the anastomosis had no additive effect on platelet deposition to downstream endothelium. In contrast, an upstream anastomosis significantly augmented platelet deposition to exposed downstream subendothelium. (Plast. Reconstr. Surg. 90: 650, 1992.)

Comparison of N-acyl phosphatidylethanolamines with different N-acyl groups as activators of glucocerebrosidase in various forms of Gaucher's disease

The acidic phospholipid requirement of the predominant particulate beta-glucosidase of mammalian spleen and liver was investigated using a series of N-acyl derivatives of dioleoyl phosphatidylethanolamine (PE). The PE, a neutral phospholipid, was converted to an acidic lipid, (N-acyl)-phosphatidylethanolamine (NAPE) by acylation of the amino group with different fatty acyl chains. Lysosomal beta-glucosidases from rat liver and spleens of controls and patients with various types of Gauchers disease were solubilized and delipidated by extraction with sodium cholate and 1-butanol. All members of the NAPE series tested were effective activators of the delipidated rat liver beta-glucosidase, and the stimulatory power of the NAPE family increased with increasing chain length of the fatty acid substitution. In contrast, dioleoyl-PE had no effect on beta-glucosidase activity. A heat-stable factor (HSF) purified from the spleen of a patient with Gauchers disease significantly increased the sensitivity of the rat liver beta-glucosidase to all of the NAPE derivatives. The maximum stimulation achieved in the presence of HSF was independent of N-acyl chain length. Compared to the potent activator, phosphatidylserine (PS), (N-acetyl)-PE and (N-linoleoyl)-PE were half as effective as activators of beta-glucosidase from control human spleen. PS stimulated the beta-glucosidase of type 1 nonneurologic Gauchers disease, but none of the NAPE compounds activated it. Neither PS nor any of the (N-acyl)-PE compounds could activate a delipidated preparation of beta-glucosidase obtained from the spleen of a neurologic case. These results indicate that although the presence of a net negative charge on a phospholipid confers upon it an ability to reconstitute beta-glucosidase activity to the normal, nonmutant enzyme, it is insufficient to permit differentiation of the various types of Gauchers disease.

The effect of microvascular anastomosis configuration on initial platelet deposition.

The propensity for platelets to bind at a native vessel anastomosis is thought to be related to subendothelial exposure, the presence of suture material, and local flow disturbances. By using an artificial microvascular graft to artificial microvascular graft anastomosis model that mimics the geometry and topography of a native microvascular anastomosis but which eliminates the endothelial and subendothelial contributions, the influence of the normal anastomotic configuration alone on initial platelet deposition was measured. Anastomotic and immediate downstream platelet deposition was not augmented by the presence of the anastomotic configuration alone. This suggests that the enhanced initial platelet deposition in the region of a native vessel microanastomosis is primarily related to the presence of injured endothelium and exposed subendothelium rather than to flow disturbances. (Plast. Reconstr. Surg. 91:522, 1993.)

Sucrose gradient analysis of phospholipid-activated β-glucosidase in type 1 and type 2 Gaucher's disease☆

Using sucrose density gradients, differences in delipidated lysosomal beta-glucosidase isolated from control spleen and spleen from patients with nonneurologic (type 1) and neurologic (type 2) Gauchers disease have been examined. The three enzymes differ in sedimentation properties as well as in their responsiveness to activation by phosphatidylserine and heat-stable factor. The control beta-glucosidase sedimented as an apparent 45,000-Da species whose activity was dependent upon the inclusion of exogenous sodium taurodeoxycholate in the assay medium. Preincubation with a mixture of phosphatidylserine and heat-stable factor converted the control enzyme to a faster-sedimenting form which exhibited considerable activity in the absence of exogenous bile salt. Spleen beta-glucosidase from a patient with type 1 Gauchers disease exhibited an apparent molecular weight of 154,000 on sucrose gradients. Like the control enzyme, the activity of this form was bile salt dependent. Upon preincubation with phosphatidylserine and heat-stable factor, beta-glucosidase from the type 1 case was also converted to a faster-sedimenting form which was more active in the absence of sodium taurodeoxycholate than in the presence of the bile salt. Spleen beta-glucosidase from the patient with type 2 Gauchers disease sedimented as a broad peak of activity in the most dense regions of the sucrose gradients, appearing to be much larger than the beta-glucosidase from either the control or the type 1 Gauchers disease patient. The activity of this large species was strongly dependent upon bile salt, and was not affected by preincubation of the enzyme with phosphatidylserine and heat-stable factor. Using the chaotropic salt, sodium thiocyanate (0.15 M), the spleen beta-glucosidase isolated from the type 1 Gauchers disease case was converted to a slower-sedimenting species. The control enzyme sedimented slightly farther into the sucrose gradients upon treatment with the NaSCN. Thiocyanate treatment had no effect on the spleen beta-glucosidase isolated from the case of type 2 Gauchers disease.

Vasospasm and platelet deposition in human arteries: effects of topical methylene blue.

Vasodilation of small blood vessels is controlled in part by the endothelium-derived relaxing factor (EDRF), which also inhibits platelet adhesion. Methylene blue (MB), which is occasionally applied directly to blood vessels during microsurgery to provide orientation and prevent torsion, is an irreversible inhibitor of the effects of endothelium-derived relaxing factor and may thereby augment both vasospasm and platelet responses. We have investigated the effects of the extravascular adventitial application of methylene blue on platelet deposition to human placental arteries (HPA) in the presence and absence of surgically induced vasospasm. A trend toward increased platelet deposition to human placental arteries was seen in each group but did not reach significance. The degree of platelet deposition to control human placental arteries suggests that the effects of methylene blue on platelet deposition may be dwarfed by the effects of surgical trauma and ischemia.

Sulfogalactocerebroside and bis-(monoacylglyceryl)-phosphate as activators of spleen glucocerebrosidase

Sequential extraction of human spleen membranes with sodium cholate and n-butanol removes endogenous lipids and renders glucocerebrosidase activity dependent upon exogenous acidic lipids (e.g., phosphatidylserine, gangliosides) and a heat-stable activator protein (HSF). In the present report, we show that two previously untested lysosomal acidic lipids, namely sulfogalactocerebroside and bis-(monoacylglyceryl)-phosphate (BMP), also activate normal human glucocerebrosidase. In addition, sulfogalactocerebroside also markedly enhanced the activity of glucocerebrosidase isolated from a patient with type 1 (non-neuronopathic) Gauchers disease, resulting in a specific activity which was 60-80% that of control glucocerebrosidase. Furthermore, when the sulfolipid was used as the activator, glucocerebrosidase from the type 1 patient was 30 times more active than the corresponding glucocerebrosidase from a person with type 2 (neuronopathic) Gauchers disease. In contrast, the two BMPs, one rich in C26 saturated fatty acid and another rich in C18 unsaturated fatty acids, were relatively poor activators of both mutant glucocerebrosidases while providing excellent reconstitution of control activity.

Quantitation of platelet deposition on human arteries: Assessment of the disparity between results obtained with 111indium (111in)-labelling versus scanning electron microscopy

Quantitation techniques for measuring platelet deposition (PD) to vessel surfaces are important to an understanding of thrombogenesis. In previous studies, scanning electron microscopy (SEM) has been shown to indicate a lower extent of PD than platelet 111In-scintigraphy. Part of this disparity may be explained by nonspecific binding of 111In to the vessel surface during perfusion, or loss of adherent 111In-labelled platelets by lysis or dissociation from the surface during specimen preparation for SEM. To assess whether these independent processes occur, we used a previously described human placental artery (HPA) perfusion model to quantify vessel 111In retention. Of the total 111In that bound to the vessel surface during perfusion, 77 +/- 42% (N = 9) was platelet associated 111In (111In-labelled platelets) and 23 +/- 19% (N = 9) was non-platelet associated 111In (nonspecific binding). After specimen fixation, 67 +/- 32% (N = 9) of the initial total surface 111In remained. This decrease is due to dissociation of both adherent 111In-labelled platelets, and nonplatelet associated 111In. After fixation, 57 +/- 34% (N = 9) of the initial total surface 111In remained as 111In-labelled platelets and 10 +/- 13% (N = 9) remained as nonplatelet associated 111In. Fixation caused no measurable lysis of platelets. These data suggest that PD may be overestimated by 111In-scintigraphy because of nonspecific binding of 111In and underestimated by SEM because of dissociation of adherent platelets during specimen preparation for SEM.

Prostacyclin production at the human microvascular anastomosis : Its effect on initial platelet deposition

&NA; The human microvascular anastomosis represents a localized environment with strongly thrombotic tendencies. In previous studies, an increase in initial platelet deposition at a human ex vivo anastomosis was measured. It is postulated that this increase in anastomotic platelet deposition was due to a reduction in anastomotic prostacyclin production as a consequence of local endothelial cell injury or loss. Instead, in this study, an increase in anastomotic prostacyclin production over unsutured controls (control 1093 ± 222 pg/ml of 6‐keto prostaglandin F (PGF) 1‐alpha, n = 21; anastomosis 2494 ± 414, n = 21, mean ± 1 SEM, p = 0.005) is demonstrated. Anastomotic prostacyclin production was augmented by addition of arachidonic acid (0.1 mM) (39,000 ± 11,300 pg/ml of 6‐keto PGF 1‐alpha, n = 7, p < 0.01) and suppressed by the preincubation of vessel segments with aspirin in a dose‐dependent fashion (1 mM) (83 ± 22 pg/ml of 6‐keto PGF 1‐alpha, n = 21, p < 0.001); aspirin (0.1 mM) (312 ± 56 pg/ml of 6‐keto PGF 1‐alpha, n = 7, p < 0.001). In further studies using a perfusion apparatus of human blood pumped through human placental artery segments, suppression of prostacyclin production did not augment initial platelet deposition (control anastomosis 4.9 ± 2.2 × 106 platelets per cm2, aspirin treatment 6.0 ± 2.8 × 106 platelets per cm2, n = 5, mean ± 1 SEM, p > 0.05). Suppression of platelet function with aspirin (0.1 mM) also did not decrease initial platelet deposition onto the anastomosis (5.8 ± 2.8 × 106 platelets per cm2, n = 4, p > 0.05. In this model system, initial platelet deposition at the anastomosis may not be dependent upon cyclooxygenase pathways. (Plast. Reconstr. Surg. 97: 784, 1996.)

The Effects of Acidic Lipids and Heat-Stable Factor on the Physical-Chemical and Kinetic Properties of Glucocerebrosidase

Glucocerebrosidase (glucocerebroside:s-glucosidase, E.C.3.2.1.45) is a lysosomal enzyme whose deficiency is responsible for the sphingolipidosis, Gaucher’s disease1,2; it catalyzes the following reaction: