Weight-dependent Manner Research Articles

Preparation and characterisation of calcium phosphate–hyaluronic acid nanocomposite bone cement

In this study, calcium phosphate cements (CPCs) were prepared by using hyaluronic acid (HA) solutions with two different molecular weights. The physical, physicochemical and structural properties of these cements were characterised and compared with those of conventional CPC as control group. Results revealed that the long setting time and the low compressive strength of the CPC could be improved drastically by using HA in the cement composition, in a molecular weight dependent manner. The HA polymer has also a promising effect on cement injectability, which is more considerable in high molecular weight HA. The X-ray diffraction patterns of set cements showed that, in both control group and HA containing cements, primary reactant components were completely converted to nanostructured apatite after soaking in simulated body fluid. This study suggests that calcium phosphate nanocomposite cement obtained by HA solution can be successfully used as injectable bone filler material.

Regulation of valvular interstitial cell phenotype and function by hyaluronic acid in 2-D and 3-D culture environments

Disruption of the extracellular matrix (ECM) is frequently found in calcific aortic valve disease (CAVD), yet the role of ECM components in valvular interstitial cell (VIC) function and dysfunction remains poorly understood. This study examines the contributions of exogenous and endogenous hyaluronic acid (HA), in both two-dimensional (2-D) and 3-D environments, in regulating the phenotype and calcification of VICs. VIC calcification was first assessed in a 2-D setting in which the cells were exposed to different molecular weights of exogenous HA presented in either an immobilized or soluble form. Delivery of HA suppressed nodule formation in a molecular weight-dependent manner, while blocking VIC recognition of HA via an antibody to CD44 abolished these nodule-suppressive effects and stimulated other hallmarks of valvular dysfunction. These 2-D results were then validated in a more physiologically-relevant setting, using an approach that allowed the characterization of VIC phenotype in response to HA alterations in the native 3-D environment. In this approach, leaflet organ cultures were analyzed following treatment with anti-CD44 or with hyaluronidase to specifically remove HA. Disruption of VIC–HA interactions upregulated markers of VIC disease and induced leaflet mineralization. Similarly, HA-deficient leaflets exhibited numerous hallmarks of CAVD, including increased VIC proliferation, apoptosis, increased expression of disease-related markers, and mineralization. These findings suggest that VIC–HA interactions are crucial in maintaining a healthy VIC phenotype. Identification ECM components that can regulate VIC phenotype and function has significant implications for understanding native valve disease, investigating possible treatments, and designing new biomaterials for valve tissue engineering.

Calcification of cartilage formed in vitro on calcium polyphosphate bone substitutes is regulated by inorganic polyphosphate

A major challenge to the successful clinical application of bioengineered cartilage remains its integration to surrounding tissues upon implantation. One way to address this consists of generating biphasic constructs composed of articular cartilage formed in vitro on the top surface and integrated with the porous sub-surface of a bone substitute material - in the case of this study, calcium polyphosphate (CPP). To improve the mechanical integrity of the cartilage-bone substitute interface, attempts have been made to generate a zone of calcified cartilage (ZCC) within the CPP-cartilage interface, thereby mimicking the native joint architecture. The purpose of this work was to establish the effects of the degradation products of CPP on cartilage calcification in order to explain the observed positioning of a ZCC away from the interface junction. It was determined that polyphosphate released from the CPP accumulates within in vitro-grown cartilage and inhibits cartilage calcification in a concentration and chain length (i.e. molecular weight) dependent manner. It was found that this effect is transient as chondrocytes express exopolyphosphatases which hydrolyze polyphosphate to release orthophosphate. Hence, the generation of biphasic constructs with a properly located ZCC will require tailoring of CPP substrates with lower degradation rates or the upregulation of exopolyphosphatases by chondrocytes.

Carboxylation of Osteocalcin Affects Its Association With Metabolic Parameters in Healthy Children

OBJECTIVEOsteocalcin (OC), a bone-derived protein, was recently shown to regulate metabolic pathways in mice. Undercarboxylated OC (ucOC), but not carboxylated OC (cOC), increases adiponectin and insulin secretion. It is unclear if carboxylation of OC affects its association with metabolic parameters in humans.RESEARCH DESIGN AND METHODSThe associations between ucOC, cOC, total and high-molecular-weight (HMW) adiponectin, and insulin secretion (homeostasis model assessment [HOMA]-β) were investigated in a population-based sample of healthy prepubertal children (n = 103; 49 boys and 54 girls).RESULTSWeight-dependent associations were observed between the different forms of OC and metabolic parameters. Higher cOC was related to lower HMW adiponectin (with a stronger association in leaner children; P < 0.001). Higher ucOC-to-cOC ratio was associated with higher HOMA-β (P < 0.01) in leaner children and associated with higher HMW adiponectin (P < 0.001) in heavier children.CONCLUSIONSIn a weight-dependent manner, cOC and the proportion of ucOC are differentially related to HMW adiponectin and insulin secretion in healthy children.

Body mass index is associated with the development of acute respiratory distress syndrome

Background:The relationship between body mass index (BMI) and development of acute respiratory distress syndrome (ARDS) is unknown.Methods:A cohort study of critically ill patients at risk for ARDS was carried out....

Mechanism of low-molecular-weight heparin reversal by platelet factor 4

Low-molecular-weight heparins (LMWH) exert their anticoagulant effect by accelerating anti-thrombin (AT) inactivation of factor Xa (fXa) and thrombin. To address the hypothesis of a calcium-dependent template mechanism in LMWH activity, we compared the ability of the heparin neutralising agent Platelet Factor 4 (PF4) to inhibit various therapeutic LMWH in a kinetic assay. Neutralization coefficients by PF4 and apparent affinities of PF4 for various LMWH increased in a molecular weight-dependent manner. Protamine sulphate neutralized heparin via a non-specific mechanism. EDTA abolished the calcium-dependent acceleration of the fXa-AT reaction, indicating that the bridging mechanism contributed significantly to LMWH activity. Within a low range of LMWH concentration (< 0.2 U/ml), excess AT over PF4 (4:1) had no effect on PF4 activity, indicating that PF4 and AT binding to heparin were independent of each other. Instead, increasing enzyme concentration reversed the negative effect of heparin- bound AT on PF4-dependent neutralization. Inhibition of thrombin by heparin was also neutralized by PF4, albeit to a higher extent than the fXa-AT reaction. Altogether, these results suggested that an interaction of PF4 with protease mediated the association of PF4 to the heparin chain. We propose that PF4 participates in the anti-fXa dependence of LMWH due to its major effect on the anti-thrombin activity of LMWH and that inhibition of fXa via the template mechanism plays an essential role in LMWH activity and pharmacokinetics, because PF4 specifically targets this mechanism.

RGD-Substituted High Molecular Weight Hyper-Branched Polyglycerols (HPG) Are Effective Platelet Inhibitors.

Peptides containing Arg-Gly-Asp (RGD) sequences are known to bind to integrins which mediate cell adhesion and therefore have been utilized in applications such as antithrombotics and tissue engineering. Although RGD and related peptides show promise, their unfavourable pharmacokinetic profiles and susceptibility to in vivo proteolysis hinder their clinical usefulness. Peptide-polymer conjugates can address one of these challenges by extending the peptide's residence in plasma. Hyperbranched polyglycerols (HPGs) are biocompatible polyether polyols with very long plasma circulation half-lives (t1/2 ∼ 60h) and as such are ideally suited to be carriers in such conjugates. HPGs of three different molecular weights were conjugated with RGD peptides at various substitution levels. Some of the terminal hydroxyl groups of polyglycerols were converted to vinyl sulfone groups which were subsequently utilized to couple cysteine terminated RGD peptides. The following conjugates were made: 515 kDa with substitution levels of 100:1, 500:1 and 1000:1; 100 kDa with substitution levels of 100:1 and 1000:1; and 3 kDa with substitution levels of 1:1, and 10:1. RGD-coupled HPG inhibited fibrinogen binding to platelet glycoprotein IIb-IIIa as detected by flow cytometry using anti-fibrinogen. Compared to free RGD (Ic50 = 5 x 10-5 M), inhibition of fibrinogen binding increased with increasing HPG molecular weight and increasing RGD-substitution for the high MW conjugates: Ic50 for the 515 kDa conjugates were 6.3 x 10−8 M for the 100:1 substitution, 4.7 x 10-8 M for the 500:1 substitution, and 4.1 x 10−8 M for 1000:1 substitution; Ic50 for the 100 kDa conjugates were 7.0 x 10−7 M for the 100:1 substitution and 1.2 x 10−7 M for the 1000:1 substitution. Ic50 for the 3 kDa conjugates were 2 x 10−5 M for the 1:1 substitution, 2 x 10−4 M for 10:1 substitution. Similarly, platelet function, as demonstrated by MnCl2-initated aggregation was inhibited in a dose- and molecular weight-dependent manner by HPG-RGD conjugates. Platelet aggregate formation and aggregate size were confirmed by microscopy. However, unsubstituted HPG had no effect on platelet fibrinogen binding and neither conjugated nor unconjugated HPG increased platelet CD62 surface expression. Trypsin digestion (but not SBTI-treated trypsin) removed the inhibitory activity of the conjugates and thus confirmed that both fibrinogen binding and aggregation-inhibition were dependent on the RGD peptide sequence. Such multi-determinant peptide-HPG conjugates represent a new approach to the development of antithrombotic drugs.

Hydrogel nerve conduits produced from alginate/chitosan complexes

Nerve conduits (NCs) represent a promising alternative to conventional treatments for peripheral nerve repair. Materials for NC production should be biodegradable, possess adequate mechanical properties, and allow for exchange of nutrients. To this aim, we developed biodegradable NC made of a hydrogel that consisted of the oppositely charged polysaccharides alginate and chitosan. Swelling and permeation studies, as well as rheological measurements, served to characterize the NC. The alginate/chitosan NC showed high water uptake (84% w/w) and permitted permeation of fluorescent-labeled dextrans in a molecular weight dependent manner. The NC fulfilled the mechanical specifications without further crosslinking. The soft NC can be expected to preclude nerve compression (storage modulus of about 40 kPa), but possess sufficient mechanical strength. In combination with remarkable tear resistance, the NC affords easy surgical handling.

182 INTERACTION OF MICROTUBULES AND MICROFILAMENTS WITH DISTRIBUTION OF MITOCHONDRIA IN TWO-CELL HAMSTER EMBRYOS

During early development, mitochondrial distribution in the ooplasm is dramatically changed. In mice and pigs, it has been reported that translocation of mitochondria is mediated by microtubules, but not by microfilaments. However, the study using hamster embryos suggested that microfilaments played a role in the mitochondrial distribution. In this study, the role of microtubules and microfilaments on distribution of mitochondria was evaluated in 2-cell hamster embryos. Female golden hamsters were superovulated on the day of post-estrus discharge by PMSG in a weight-dependent manner [Mackiernan and Bavister 2000 Hum. Reprod. 15, 157–164], and mated with males in the evening 3 days later. Two-cell embryos were collected from the oviducts at 0900–1000 on Day 2 of pregnancy. Embryos were cultured in HECM-10 medium with or without nocodazole (a microtubule assembly inhibitor), and/or cytochalasin D (a microfilament assembly inhibitor) for 8–10 h in a humidified atmosphere of 10% CO2, 5% O2, and 85% N2 at 37.5&amp;deg;C. After incubation of embryos with or without inhibitors, mitochondria were stained with rhodamine 123. Some of them were stained for microtubules, microfilaments, and DNA, as reported previously (Suzuki et al. 1999 Biol. Reprod. 61, 521–526). In 2-cell hamster embryos (controls, n = 44), most mitochondria were accumulated at the perinuclear region, while little mitochondria were noted in the cell cortex. Microtubules were found around the nuclei, and distribution of the mitochondria was somewhat correlated with that of the microtubules. In contrast, microfilaments were located in a high density just under the cell membrane. After nocodazole treatment (n = 22), mitochondria had extended into the subcortical (intermediate) region in various degrees, where mitochondria were aggregated into large masses. After cytochalasin D treatment (n = 17), distributional density of mitochondria at the peripheral region was decreased, and mitochondria moved back around the nucleus. After treatment by both inhibitors (n = 21), a distribution pattern of mitochondria was almost similar to that observed after cytochalasin D treatment. The present results suggest that the distribution of mitochondria at the perinuclear region is mediated by microtubules, whereas the movement of mitochondria to the cell cortex is regulated by microfilaments. Microfilaments seemed to play a role as the anchor of mitochondria at the cell cortex.

Multi-layered nanoball as high performance permselective membrane

Multi-layered structure of the core-polymerized polymeric micelle carrying aldehyde groups (aldehyde-nanoball) and polyallylamine (PAA) was prepared on a porous polymer membrane by the layer-by-layer (LBL) coating, followed by reductive amination. Aldehyde-nanoball with diameter of 40 nm was made from a core-shell type polymeric micelle of acetal-poly(ethylene glycol)/polylactide-methacryloyl block copolymer (acetal-PEG/PLA-methacryloyl) by a gentle acid treatment to convert acetal into aldehyde group, followed by the polymerization of the methacryloyl group in the core. Permselectivity of hydrophilic macromolecules (dextran, myoglobin, albumin) through the nanoball-coated membrane was evaluated by the use of two-chamber cell. With an increasing number of the coating layers, permeation of the macromolecules was reduced in the molecular weight-dependent manner. Especially, in the case of protein molecules as solutes, a definite cut-off molecular weight was justified by changing the number of the coating layer. The multi-layered coating of nanoballs developed here is expected to be useful as a high performance separation membrane for proteinous compounds.

RUNX2 alleles associated with BMD in Scottish women; interaction of RUNX2 alleles with menopausal status and body mass index

Bone mineral density (BMD) is influenced by both environmental and genetic factors. We previously reported the association of the RUNX2 A allele with increased bone mineral density (BMD) and protection against a common form of osteoporotic fracture within a Geelong population. We genotyped 991 women from a Scottish cohort to decipher the role of RUNX2 alleles in regulating BMD. The alleles of RUNX2 within the glutamine–alanine repeat were determined by MspA1I restriction digest. Allele frequencies estimated from Scottish cohort were G allele, 0.87 ± 0.01; A allele, 0.08 ± 0.01; and 11Ala alanine deletion allele, 0.05 ± 0.01. Analysis of covariance (ANCOVA) was used to adjust for the covariates weight and age for BMD at the femoral neck (FN). The A allele was associated with higher FN BMD ( P = 0.035) within a postmenopausal subgroup of the population ( n = 312). The effect of RUNX2 A alleles increased with increasing weight; A alleles were associated with FN BMD in those above the median BMI (BMI > 25), while no association was observed in thin/normal (BMI ≤ 25) postmenopausal women. Glutamine variants and an alanine insertion were identified within the group. These data suggest that the RUNX2 alleles are associated with BMD in a menopause- and weight-dependent manner.

Enhanced cellular uptake of a triplex-forming oligonucleotide by nanoparticle formation in the presence of polypropylenimine dendrimers

We used polypropylenimine dendrimers for delivering a 31 nt triplex-forming oligonucleotide (ODN) in breast, prostate and ovarian cancer cell lines, using 32P-labeled ODN. Dendrimers enhanced the uptake of ODN by approximately 14-fold in MDA-MB-231 breast cancer cells, compared with control ODN uptake. Dendrimers exerted their effect in a concentration- and molecular weight-dependent manner, with generation 4 (G-4) dendrimer having maximum efficacy. A similar increase in ODN uptake was found with MCF-7 and SK-BR-3 (breast), LNCaP (prostate) and SK-OV-3 (ovarian) cancer cells. The dendrimers had no significant effect on cell viability at concentrations at which maximum ODN uptake occurred. [3H]Thymidine incorporation showed that complexing the ODN with G-4 significantly increased the growth-inhibitory effect of the ODN. Western blot analysis showed a significant 65% reduction of c-myc protein level in ODN-G-4 treated cells compared with that of ODN-treated/control cells. Gel electrophoretic analysis showed that ODN remained intact in cells even after 48 h of treatment. The hydrodynamic radii of nanoparticles formed from ODN in the presence of the dendrimers were in the range of 130-280 nm, as determined by dynamic laser light scattering. Taken together, our results indicate that polypropylenimine dendrimers might be useful vehicles for delivering therapeutic oligonucleotides in cancer cells.

Dose-Dependent and Preterm- Accentuated Diaplacental Transport of Nutritive Allergens in vitro

Objective: The mechanisms by which nutritive allergens are transported from mother to fetus and the ensuing immunological response are incompletely understood. We investigated the role of different allergen concentrations in influencing the diaplacental allergen transport in preterm and term placentas. Method: Twenty-seven human term placentas and 12 preterm placentas were dually perfused in vitrofor up to 4 h by adding alternately two different nutritive allergens, β-lactoglobulin (BLG) or ovalbumin (OVA), at four different allergen concentrations (0.02, 0.2, 2 and 20 mg/ml) to the maternal perfusate medium. Allergen concentrations in fetal venous outflow samples collected during perfusion were measured by using specific ELISAs. Results: Perfusion of increasing allergen concentrations via the maternal circulation resulted in a concentration-dependent increase of fetal allergen uptake in all term and preterm placentas. A mean maternal-to-fetal ratio of 20,000/1 and 3,000/1 for BLG, and 40,000/1 and 5,000/1 for OVA was found in term and preterm placentas, respectively. Preterm placentas (27–36 weeks of gestation) were found to favor the diaplacental passage of nutritive allergens compared with placentas at term (>36 weeks of gestation). Conclusion: Maternal-to-fetal allergen transport occurs in a dose-dependent and molecular weight-dependent manner with clear accentuation in preterm placentas.

Enhanced ability of heparin-carrying polystyrene (HCPS) to bind to heparin-binding growth factors and to inhibit growth factor-induced endothelial cell growth.

Heparin-carrying polystyrene (HCPS) consists of low-molecular-weight heparin chains enriched in trisulfated disaccharide structures linked to a polystyrene core. In this study, the interactions between HCPSs of various molecular weights and heparin-binding growth factors, VEGF(165), FGF-2, and HGF, were compared to the interactions of the same factors with native heparin, periodate-oxidized heparin (IO(4)-heparin) and periodate-oxidized alkaline-degraded heparin (IO(4)-LMW-heparin). The binding of each growth factor to heparin-agarose beads (heparin-beads) was more strongly inhibited by HCPSs in a molecular weight-dependent manner than by native heparin or the modified heparins, indicating a stronger interaction between HCPS and these growth factors. HCPSs also inhibit heparin-binding growth factor-induced endothelial cell growth in a molecular weight-dependent manner much more strongly than the native or modified heparins. However, HCPSs did not inhibit the mitogenic activity of VEGF(121), which has a non-heparin-binding nature. Thus, HCPSs exhibit enhanced abilities to interact with each of the heparin-binding growth factors studied and to inhibit heparin-binding growth factor-induced endothelial cell proliferation in a molecular weight-dependent manner. These effects might be ascribed to the heparin-clustering effect of HCPSs.

Midlatency auditory-evoked potentials in the rat: effects of interventions that modulate arousal

The vertex-recorded P13 midlatency auditory-evoked potential in the rat shows the same characteristics as the P1 potential in the human, namely, sleep-state dependence, rapid habituation and blockade by the cholinergic antagonist scopolamine. The P13 potential appears to be generated, at least in part, by projections of the pedunculopontine nucleus, the cholinergic arm of the reticular activating system. On the other hand, the auditory cortex-recorded P7 potential appears to be of primary cortical origin. Simultaneous recordings from the vertex and the auditory cortex showed that (1) the P13 potential was suppressed by administration of the anesthetics ketamine, pentobarbital or halothane in a dose-dependent manner, but the P7 potential was not; (2) the P13 potential was suppressed by intragastric injections of ethanol in a dose-dependent manner, but the P7 potential was not; (3) the amplitude of the P13 potential was negatively correlated with blood ethanol levels; (4) both the P13 and P7 potentials were still present following injections of the neuromuscular blocker pancuronium bromide; and (5) both the P13 and P7 potentials were decreased by diffuse brain injury induced by a weight-drop device in a weight-dependent manner. These findings suggest that the P13 potential is more sensitive than the P7 potential to changes in arousal and that the P13 and P7 potentials are not of myogenic but of neural origin.

Influence of Polylysine on the Rupture of Negatively Charged Membranes

We investigate the stability and rupture kinetics of planar lipid membranes covered with electrostatically adsorbed polyelectrolytes. After black lipid membranes were formed from negatively charged lipids, polylysines (PLs) of different molecular weights (MW) were added on one or on both sides of the membrane. The adsorption of PL was detected by recording changes of the transmembrane voltage. Rupture was induced by applying short voltage pulses across the membrane. The voltage causing breakdown of the membrane gives information on its mechanical stability. Adsorption of PL on one side of the membrane leads to an asymmetric transmembrane potential, which adds to the externally applied voltage. High MW PL decreases the critical breakdown voltage of the membrane significantly but also increases the delay time between the voltage pulse and pore formation. It is further shown that PL alters the time course of pore widening in a molecular weight-dependent manner. Low MW PL-decorated membranes and undecorated membranes show a fast rupture determined by inertia. In contrast, adsorption of high MW PLs causes a dramatic decrease of the rupture velocity. In this case, the rupture velocity is determined by viscosity. An analysis of the rupture kinetics allows an estimate of the 2D viscosity.

Inhibition of pulmonary surfactant biophysical activity by cationic polyamino acids.

The purpose of this study is to investigate the interaction of cationic polyamino acids, polylysine and polyarginine, with rat pulmonary surfactant at the air/water interface. Surface pressure measurements of rat pulmonary surfactant in the presence and absence of polyamino acids were carried out in both dynamic and static modes. In dynamic cycle studies, compression and expansion of adsorbed surfactant films in the presence of the cationic polyamino acids resulted in a delayed attainment of the plateau surface pressure. In area studies of spread surfactant films at constant surface pressure, cationic polyamino acids in the subphase resulted in an increase in film area. Increased film area was also observed when a polyamino acid was injected beneath films of dipalmitoyl-phosphatidylcholine/phosphatidylglycerol. In the presence of the cationic polyamino acids, the equilibrium surface pressure (at constant film area) of pulmonary surfactant was elevated in a concentration- and molecular weight-dependent manner. These data indicate that the model cationic peptides interact with surfactant lipid, possibly electrostatically with phosphatidylglycerol. It is concluded that the surface activity of pulmonary surfactant is significantly inhibited by the presence of the polycations, possibly by the formation of a mixed lipid/polyamino acid film.

The effect of hyaluronan on interleukin-1 alpha-induced prostaglandin E2 production in human osteoarthritic synovial cells.

An in vitro study on the effects of hyaluronan (HA) on interleukin-1 alpha-induced prostaglandin E2 (PGE2) production in human osteoarthritic synovial cells indicated that PGE2 induction was suppressed by HA in a dose- and molecular weight-dependent manner.

Effects of sodium hyaluronate on the nociceptive response of rats with experimentally induced arthritis

Antinociceptive effects of sodium hyaluronate (Na-HA) were studied on the basis of improvement in the graded abnormal gait elicited by arthritis induced by intra-articular administration of monosodium urate crystal (MSU) to rats. One hour before MSU injection, intra-articular administration of a 1.0% solution of Na-HA with different molecular weights, ranging from 4.70 x 10(5) to 2.02 x 10(6) (HA-200), improved the score of abnormal gait in a molecular weight-dependent manner in the experimental arthritis model. Similarly, administrations of HA-200 at concentrations ranging from 0.1 to 1.0% prior to MSU treatment resulted in improvement of the score in abnormal gait in a dose-dependent manner. To elucidate the antinociceptive mechanisms of Na-HA, effects of pretreatment with Na-HA (1.0%) of different molecular weights on prostaglandin E2 (PGE2) and bradykinin (BK) releases in synovial fluid 3 hr after MSU injection were studied. Increases in PGE2 and BK concentration in the synovial fluid were depressed in a molecular weight-dependent manner by Na-HA (1.0%) pretreatment. These results indicate that Na-HA attenuates the nociceptive responses inflicted by the MSU-induced arthritis. Such an antinociceptive effect may be due to the inhibition of PGE2 and BK synthesis in the synovial joint of rats.

Heparin-induced platelet aggregation. II. Dose/response relationships for two low molecular weight heparin fractions (CY 216 and CY 222)

We have previously shown that unmodified heparin (bovine lung or porcine mucosal) and a low molecular weight heparin fraction, PK 10169, cause platelet aggregation in a dose and molecular weight-dependent manner. In this report, we show that two other low molecular weight heparin fractions, CY 216 and CY 222, also cause platelet aggregation in a dose and molecular weight-dependent manner. Utilizing heparin and defined fractions of CY 216 and CY 222 separated on the basis of molecular weight, we determined dose/response (D/R) relationships for each of these agents and their individual fractions. In comparison to an unmodified porcine mucosal heparin, CY 216 yielded a D/R curve that was shifted down and to the right, indicating that this agent is less potent in causing platelet aggregation. The D/R curve for CY 222, which has a lower molecular weight that CY 216, was shifted further down and to the right, indicating that it was less potent than CY 216. The D/R curves obtained with the fractions of CY 216 and CY 222 demonstrate that as the molecular weight of the fractions decrease, they become progressively less potent in causing platelet aggregation. Fractions with molecular weights of less than approximately 3,000 daltons are essentially without activity in causing platelet aggregation. Platelet aggregation studies with CY 216 and CY 222 fractions separated on the basis of affinity for antithrombin III (AT III) indicate that the platelet aggregating activity of these agents may not be related to their affinity for AT III. However, these latter results are not conclusive and need to be expanded.